Compositions for optimized RAS peptide vaccines

ABSTRACT

The present disclosure provides for methods, systems, and compositions of nucleic acid and peptide sequences. The present disclosure provides for a nucleic acid sequence encoding two or more amino acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41. The present disclosure also provides for an immunogenic peptide composition comprising two or more peptides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41. The present disclosure further provides for a nucleic acid sequence encoding one or more amino acid sequences selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65. The present disclosure additionally provides for an immunogenic peptide composition comprising one or more peptides selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction of the patent document or the patent disclosure as itappears in the U.S. Patent and Trademark Office patent file or records,but otherwise reserves any and all copyright rights.

INCORPORATION BY REFERENCE

All documents cited herein are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on Nov. 20, 2020, isnamed 2215269_00123US1_SL.txt and is 419,529 bytes in size.

TECHNICAL FIELD

The present invention relates generally to compositions, systems, andmethods of peptide vaccines. More particularly, the present inventionrelates to compositions, systems, and methods of designing peptidevaccines to treat or prevent disease optimized based on predictedpopulation immunogenicity.

BACKGROUND

The goal of a peptide vaccine is to train the immune system to recognizeand expand its capacity to engage cells that display foreign peptides toimprove the immune response to cancerous cells or pathogens. A peptidevaccine can also be administered to someone who is already diseased toincrease their immune response to a causal cancer, other diseases, orpathogen. There exists a need for compositions, systems, and methods ofpeptide vaccines based on prediction of the foreign peptides that willbe displayed at a later time to protect a host from cancer, otherdisease, or pathogen infection.

SUMMARY OF THE INVENTION

In one aspect, the invention provides for a nucleic acid sequenceencoding two or more amino acid sequences selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.

In some embodiments, the nucleic acid sequence is an immunogeniccomposition. In some embodiments, the nucleic acid sequence isadministered in a construct for expression in vivo. In some embodiments,the in vivo administration of the nucleic acid sequence is configured toproduce one or more peptides that are displayed by an HLA class Imolecule. In some embodiments, the one or more peptides is a modified orunmodified fragment of a mutated KRAS protein. In some embodiments, themutated KRAS protein is selected from the group consisting of KRAS G12D,KRAS G12V, and KRAS G12R. In some embodiments, the nucleic acid sequenceis administered in an effective amount to a subject to prevent cancer.In some embodiments, the nucleic acid sequence is administered in aneffective amount to a subject to treat cancer.

In another aspect, the invention provides for an immunogenic peptidecomposition comprising two or more peptides selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.

In some embodiments, a peptide in the immunogenic peptide composition isdisplayed by an HLA class I molecule. In some embodiments, a peptide inthe immunogenic peptide composition is a modified or unmodified fragmentof a mutated KRAS protein. In some embodiments, the mutated KRAS proteinis selected from the group consisting of KRAS G12D, KRAS G12V, and KRASG12R. In some embodiments, the immunogenic peptide composition isadministered in an effective amount to a subject to prevent cancer. Insome embodiments, the immunogenic peptide composition is administered inan effective amount to a subject to treat cancer. In some embodiments,the immunogenic peptide composition comprises at least three peptidesselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.

In another aspect, the invention provides for a nucleic acid sequenceencoding one or more amino acid sequences selected from the groupconsisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:64, and SEQ ID NO: 65.

In some embodiments, the nucleic acid sequence is an immunogeniccomposition. In some embodiments, the nucleic acid sequence isadministered in a construct for expression in vivo. In some embodiments,the in vivo administration of the nucleic acid sequence is configured toproduce one or more peptides that are displayed by an HLA class IImolecule. In some embodiments, the one or more peptides is a modifiedfragment of a mutated KRAS protein. In some embodiments, the mutatedKRAS protein is selected from the group consisting of KRAS G12D, KRASG12V, KRAS G12R, KRAS G12C, and KRAS G13D. In some embodiments, thenucleic acid sequence is administered in an effective amount to asubject to prevent cancer. In some embodiments, the nucleic acidsequence is administered in an effective amount to a subject to treatcancer.

In another aspect, the invention provides for an immunogenic peptidecomposition comprising one or more peptides selected from the groupconsisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:64, and SEQ ID NO: 65.

In some embodiments, a peptide in the immunogenic peptide composition isdisplayed by an HLA class II molecule. In some embodiments, a peptide inthe immunogenic peptide composition is a modified or unmodified fragmentof a mutated KRAS protein. In some embodiments, the mutated KRAS proteinis selected from the group consisting of KRAS G12D, KRAS G12V, KRASG12R, KRAS G12C, and KRAS G13D. In some embodiments, the immunogenicpeptide composition is administered in an effective amount to a subjectto prevent cancer. In some embodiments, the immunogenic peptidecomposition is administered in an effective amount to a subject to treatcancer. In some embodiments, the immunogenic peptide compositioncomprises at least two peptides selected from the group consisting ofSEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ IDNO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60,SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ IDNO: 65.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures depict illustrative embodiments of the invention.

FIG. 1 is a flow chart of a vaccine optimization method.

FIG. 2 is a flow chart of vaccine optimization method with seed setcompression.

FIG. 3 shows predicted population coverage for single target MHC class Ivaccines by vaccine size for KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C,and KRAS G13D targets.

FIG. 4 shows predicted population coverage for single target MHC classII vaccines by vaccine size for KRAS G12D, KRAS G12V, KRAS G12R, KRASG12C, and KRAS G13D targets.

FIG. 5 shows probabilities of disease presentations for pancreas,colon/rectum, and bronchus/lung and respective probabilities of targetpresentations for KRAS G12D, KRAS G12V, and KRAS G12R targets.

FIG. 6 is a flow chart for multiple target (combined) vaccineoptimization methods.

FIG. 7 shows predicted population coverage for pancreatic cancermultiple target (combined) MHC class I vaccines by vaccine size for KRASG12D, KRAS G12V, and KRAS G12R targets.

FIG. 8 shows predicted population coverage for pancreatic cancermultiple target (combined) MHC class II vaccines by vaccine size forKRAS G12D, KRAS G12V, and KRAS G12R targets.

FIG. 9 shows an example Python implementation of the MERGEMULTI functionfor combined vaccine design procedures.

FIG. 10 shows predicated peptide-HLA hits by vaccine size for a KRASG12V vaccine for the HLA diplotype HLA-A02:03, HLA-A11:01, HLA-B55:02,HLA-B58:01, HLA C03:02, HLA-C03:03.

DETAILED DESCRIPTION

In some embodiments, the disclosure provides for peptide vaccines thatincorporate peptide sequences that will be displayed by MajorHistocompatibility Complex (MHC) molecules on cells and train the immunesystem to recognize cancer or pathogen diseased cells. In someembodiments, a peptide vaccine is a composition that consists of one ormore peptides. In some embodiments, a peptide vaccine is an mRNA or DNAconstruct administered for expression in vivo that encodes for one ormore peptides.

Peptide display by an MHC molecule is necessary, but not sufficient, fora peptide to be immunogenic and cause the recognition of the resultingpeptide-MHC complex by an individual's T cells to trigger T cellactivation, expansion, and immune memory. In some embodiments,experimental data from assays such as the ELISPOT (Slota et al., 2011)or the Multiplex Identification of Antigen-Specific T Cell ReceptorsUsing a Combination of Immune Assays and Immune Receptor Sequencing(MIRA) assay (Klinger et al., 2015) can be combined with machinelearning based predictions of peptide display by an MHC molecule. Insome embodiments, the MHCflurry or NetMHCpan computational methods(known in the art) are used to predict MHC class I display of a peptideby an HLA allele (see Table 1). In some embodiments, the NetMHCIIpancomputational method (known in the art) is used to predict MHC class IIdisplay of a peptide by an HLA allele (see Table 2).

A peptide is displayed by an MHC molecule when it binds within thegroove of the MHC molecule and is transported to the cell surface whereit can be recognized by a T cell receptor. In some embodiments, apeptide that is part of the normal proteome in a healthy individual is aself-peptide, and a peptide that is not part of the normal proteome is aforeign peptide. Foreign peptides can be generated by mutations innormal self-proteins in tumor cells that create epitopes calledneoantigens, or by pathogenic infections. In some embodiments, aneoantigen is any subsequence of a human protein, where the subsequencecontains one or more altered amino acids or protein modifications thatdo not appear in a healthy individual.

For example, KRAS gene mutations are the most frequently mutatedoncogenes in cancer, but they have been very difficult to treat withsmall molecule therapeutics. The KRAS protein is part of a signalingpathway that controls cellular growth, and point mutations in theprotein can cause constitutive pathway activation and uncontrolled cellgrowth. Single amino acid KRAS mutations result in minor changes inprotein structure, making it difficult to engineer small molecule drugsthat recognize a mutant specific binding pocket and inactivate KRASsignaling. KRAS oncogenic mutations include the mutation of position 12from glycine to aspartic acid (G12D), glycine to valine (G12V), glycineto arginine (G12R), or glycine to cystine (G12C); or the mutation ofposition 13 from glycine to aspartic acid (G13D). The correspondingforeign peptides contain these mutations.

A challenge for the design of peptide vaccines is the diversity of humanMHC alleles that each have specific preferences for the peptidesequences they will display. The Human Leukocyte Antigen (HLA) loci,located within the MHC, encode the HLA class I and class II molecules.There are three classical class I loci (HLA-A, HLA-B, and HLA-C) andthree loci that encode class II molecules (HLA-DR, HLA-DQ, and HLA-DP).An individual's HLA type describes the alleles they carry at each ofthese loci. Peptides of length of between about 8 and about 11 residuescan bind to HLA class I (or MHC class I) molecules whereas those oflength of between about 13 and about 25 bind to HLA class II (or MHCclass II) molecules (Rist et al., 2013; Chicz et al., 1992). Humanpopulations that originate from different geographies have differingfrequencies of HLA alleles, and these populations exhibit linkagedisequilibrium between HLA loci that result in population specifichaplotype frequencies. In some embodiments, methods are disclosed forcreating effective vaccines that includes consideration of the HLAallelic frequency in the target population, as well as linkagedisequilibrium between HLA genes to achieve a set of peptides that islikely to be robustly displayed.

The present disclosure provides for compositions, systems, and methodsof vaccine designs that produce immunity to multiple targets. In someembodiments, a target is a neoantigen protein sequence, a pathogenproteome, or any other undesired protein sequence that is non-self andis expected to be bound and displayed by an MHC molecule. When a targetis present in an individual it may result in multiple peptide sequencesthat are displayed by a variety of HLA alleles. Therefore, in thisdisclosure, “foreign peptide” refers to an amino acid sequence encodinga fragment of a target protein/peptide (or a full protein/peptide), thetarget protein/peptide consisting of: a neoantigen protein, a pathogenproteome, or any other undesired protein that is non-self and isexpected to be bound and displayed by an MHC molecule.

In some embodiments, peptide-MHC immunogenicity data or computationalpredictions of peptide-MHC immunogenicity can be included and combinedwith scores for peptide display in the methods disclosed herein. One wayof combining the scores is using immunogenicity data for peptidesassayed for immunogenicity in diseased or vaccinated individuals, andassigning peptides to the HLA allele that displayed them in theindividual by choosing the HLA allele that computational methods predicthas the highest likelihood of display. For peptides that are notexperimentally assayed, computational predictions of display can beused.

Since immunogenicity may vary from individual to individual, one methodto increase the probability of vaccine efficacy is to use a diverse setof foreign peptides (e.g., at least two peptides) to increase thechances that some subset of them will be immunogenic in a givenindividual. Prior research using mouse models has shown that most MHCdisplayed peptides are immunogenic, but immunogenicity varies fromindividual to individual as described in Croft et al. (2019). In someembodiments, experimental peptide-HLA immunogenicity data are used todetermine which foreign peptides and their modifications will beeffective immunogens in a vaccine.

Considerations for the design of peptide vaccines are outlined in Liu etal., Cell Systems 11, Issue 2, p. 131-146 (Liu et al., 2020) and (Liu etal., 2020b) which are incorporated by reference herein.

Certain foreign peptides may not bind with high affinity to a wide rangeof HLA molecules. To increase the binding of foreign peptides to HLAmolecules, their amino acid composition can be altered to change one ormore anchor residues or other residues. Anchor residues are amino acidsthat interact with an HLA molecule and have the largest influence on theaffinity of a peptide for an HLA molecule. Peptides with altered anchorresidues are called heteroclitic peptides. In some embodiments,heteroclitic peptides include foreign peptides with residuemodifications at non-anchor positions. In some embodiments, heterocliticpeptides include foreign peptides with residue modifications thatinclude unnatural amino acids and amino acid derivatives. Modificationsto create heteroclitic peptides can improve the binding of peptides toboth MHC class I and MHC class II molecules, and the modificationsrequired can be both peptide and MHC class specific. Since peptideanchor residues face the MHC molecule groove, they are less visible thanother peptide residues to T cell receptors. Thus, heteroclitic peptideshave been observed to induce a T cell response where the stimulated Tcells also respond to unmodified peptides. It has been observed that theuse of heteroclitic peptides in a vaccine can improve a vaccine'seffectiveness (Zirlik et al., 2006). In some embodiments, theimmunogenicity of heteroclitic peptides are experimentally determinedand their ability to activate T cells that also recognize thecorresponding base (also called seed) peptide of the heterocliticpeptide is performed as is known in the art. In some embodiments, theseassays of the immunogenicity and cross-reactivity of heterocliticpeptides are performed when the heteroclitic peptides are displayed byspecific HLA alleles.

Peptide Vaccines to Induce Immunity to One or More Targets

In some embodiments, a method is provided for formulating peptidevaccines using a single vaccine design for a one or more targets. Insome embodiments, a single target is a foreign protein with a specificmutation (e.g., KRAS G12D). In some embodiments, multiple targets can beused (e.g. both KRAS G12D and KRAS G13D).

In some embodiments, the method includes extracting peptides toconstruct a candidate set from all target proteome sequences (e.g.,entire KRAS G12D protein) as described in Liu et al. (2020). FIGS. 1 and2 depict flow charts for example vaccine design methods that can be usedfor MHC class I or MHC class II vaccine design. In some embodiments,extracted foreign peptides are of amino acid length of between about 8and about 10 (e.g., for MHC class I binding (Rist et al., 2013)). Insome embodiments, the extracted foreign peptides presented by MHC classI molecules are longer than 10 amino acid residues, such as 11 residues(Trolle et al., 2016). In some embodiments, extracted foreign peptidesare of length between about 13 and about 25 (e.g., for class II binding(Chicz et al., 1992)). In some embodiments, sliding windows of varioussize ranges described herein are used over the entire proteome. In someembodiments, other foreign peptide lengths for MHC class I and class IIsliding windows can be utilized. In some embodiments, computationalpredictions of proteasomal cleavage are used to filter or selectpeptides in the candidate set. One computational method for predictingproteasomal cleavage is described by Nielsen et al. (2005). In someembodiments, peptide mutation rates, glycosylation, cleavage sites, orother criteria can be used to filter peptides as described in Liu et al.(2020).

As shown in FIGS. 1-2, in some embodiments, the next step of the methodincludes scoring the foreign peptides in the candidate set for bindingto all considered HLA alleles as described in Liu et al. (2020) and Liuet al. (2020b). Scoring can be accomplished for human HLA molecules,mouse H-2 molecules, swine SLA molecules, or MHC molecules of anyspecies for which prediction algorithms are available or can bedeveloped. Thus, vaccines targeted at non-human species can be designedwith the method. Scoring metrics can include the affinity for a foreignpeptide to an HLA allele in nanomolar, eluted ligand, presentation, andother scores that can be expressed as percentile rank or any othermetric. The candidate set may be further filtered to exclude peptideswhose predicted binding cores do not contain a particular pathogenic orneoantigen target residue of interest or whose predicted binding corescontain the target residue in an anchor position. The candidate set mayalso be filtered for foreign peptides of specific lengths, such aslength 9 for MHC class I, for example. In some embodiments, scoring offoreign peptides is accomplished with experimental data or a combinationof experimental data and computational prediction methods.

The criteria used for scoring peptide-HLA binding during the scoringprocedure can accommodate different goals during the candidateidentification and vaccine design phases. For example, a foreign peptidewith peptide-HLA binding affinities of 500 nM may be displayed by anindividual that is diseased, but at a lower frequency than a foreignpeptide with a 50 nM peptide-HLA binding affinity. Thus, during thescoring of a candidate set to qualify potential immune system targets,500 nM or other less constrained affinity criteria than 50 nM may beutilized. During the combinatorial design phase of a vaccine, a moreconstrained affinity criteria may be used, such a 50 nM, to increase theprobability that a vaccine peptide will be found and displayed by HLAmolecules. In some embodiments, peptides are selected that havepeptide-HLA binding affinities of between about 50 nM and about 500 nM.Alternatively, combined models that incorporate peptide immunogenicitycan be used to qualify foreign peptides for improvement and score theirmodified versions for vaccine inclusion. In some embodiments,experimental observations of the immunogenicity of peptides in thecontext of their display by HLA alleles can be used to score peptidesfor vaccine inclusion. In some embodiments, computational predictions ofthe immunogenicity of a peptide in the context of display by HLA allelescan used for scoring such as the methods of Ogishi et al. (2019).

In some embodiments, the method further includes running theOptiVax-Robust algorithm as described in Liu et al. (2020) using the HLAhaplotype frequencies of a population on the scored candidate set toconstruct a seed set (also referred to as base set herein) of foreignpeptides (FIG. 2). In some embodiments, HLA diplotype frequencies can beprovided to OptiVax. OptiVax-Robust includes algorithms to eliminatepeptide redundancy that arises from the sliding window approach withvarying window sizes, but other redundancy elimination measures can beused to enforce minimum edit distance constraints between foreignpeptides in the candidate set. The size of the seed set is determined bya point of diminishing returns of population coverage as a function ofthe number of foreign peptides in the seed set. Other criteria can alsobe used, including a minimum number of vaccine foreign peptides, maximumnumber of vaccine foreign peptides, and desired predicted populationcoverage. One alternative criterion is a minimum number of expectedpeptide-HLA hits in each individual, where a peptide-HLA hit is thepotential immunogenic display of a peptide by a single HLA allele asdescribed as in Liu et al. (2020b). In alternate embodiments, the methodfurther includes running the OptiVax-Unlinked algorithm as described inLiu et al. (2020) instead of OptiVax-Robust.

The OptiVax-Robust method uses binary predictions of foreign peptidebinding to HLA alleles, and these binary predictions can be generated asdescribed in Liu et al. (2020). The OptiVax-Unlinked method uses theprobability of foreign peptide binding to HLA alleles and can begenerated as described in Liu et al. (2020). Either method can be usedfor the purposes described herein, and thus we will the term “OptiVax”refers to either the Robust or Unlinked method. In some embodiments, theobserved probability of peptide-HLA immunogenicity in experimentalassays can be used as the probability of peptide-HLA binding inEvalVax-Unlinked and OptiVax-Unlinked. In some embodiments, the HLAhaplotype or HLA allele frequencies of a population provided to OptiVaxfor vaccine design describe the world's population. In alternativeembodiments, the HLA haplotype or HLA allele frequencies of a populationprovided to OptiVax for vaccine design are specific to a geographicregion. In alternative embodiments, the HLA haplotype or HLA allelefrequencies of a population provided to OptiVax for vaccine design arespecific to an ancestry. In alternative embodiments, the HLA haplotypeor HLA allele frequencies of a population provided to OptiVax forvaccine design are specific to a race. In alternative embodiments, theHLA haplotype or HLA allele frequencies of a population provided toOptiVax for vaccine design are specific to individuals with risk factorssuch as genetic indicators of risk, age, exposure to chemicals, alcoholuse, chronic inflammation, diet, hormones, immunosuppression, infectiousagents, obesity, radiation, sunlight, or tobacco use. In alternativeembodiments, the HLA haplotype or HLA allele frequencies of a populationprovided to OptiVax for vaccine design are specific to individuals thatcarry certain HLA alleles. In alternative embodiments, the HLAdiplotypes provided to OptiVax for vaccine design describe a singleindividual, and are used to design an individualized vaccine.

In some embodiments, the seed set of foreign peptides that results fromOptiVax application to the candidate set of target peptides describes aset of unmodified foreign peptides that represent a possible compactvaccine design (Seed Set in FIG. 2). In some embodiments, the seed setis based upon filtering candidate peptides by predicted or observedaffinity or immunogenicity with respect to HLA molecules (Seed Set inFIG. 1). However, to improve the display of the foreign peptides in awide range of HLA haplotypes as possible, some embodiments includemodifications of the seed (or base) set. In some embodiments,experimental assays can be used to ensure that a modified seed (or base)peptide activates T cells that also recognize the seed peptide.

For a given foreign peptide, the optimal anchor residue selection maydepend upon the HLA allele that is binding to and displaying the foreignpeptide and the class of the HLA allele (MHC class I or class II). Aseed peptide set can become an expanded set by including anchor residuemodified peptides of either MHC class I or II peptides (FIGS. 1-2).Thus, one aspect of vaccine design is considering how to select alimited set of heteroclitic peptides that derive from the same foreignpeptide for vaccine inclusion given that different heteroclitic peptideswill have different and potentially overlapping population coverages.

In some embodiments, all possible anchor modifications for each base setforeign peptide are considered. There are two anchor residues inpeptides bound by MHC class I molecules, typically at positions 2 and 9for 9-mer peptides. At each anchor position, 20 possible amino acids areattempted in order to select the best heteroclitic peptides. Thus, forMHC class I binding, 400 (i.e., 20 amino acids by 2 positions=20²) minus1 heteroclitic peptides are generated for each base foreign peptide.There are four anchor residues in peptides bound by MHC class IImolecules, typically at positions 1, 4, 6, and 9 of the 9-mer bindingcore. Thus, for MHC class II binding there are 160,000 (i.e., 20 aminoacids by 4 positions=20⁴) minus 1 heteroclitic peptides generated foreach base foreign peptide. Other methods, including Bayesianoptimization, can be used to select optimal anchor residues to createheteroclitic peptides from each seed (or base) set peptide. Othermethods are presented in “Machine learning optimization of peptides forpresentation by class II MHCs” by Dai et al. (2020), incorporated in itsentirety herein. In some embodiments, the anchor positions aredetermined by the HLA allele that presents a peptide, and thus the setof heteroclitic peptides includes for each set of HLA specific anchorpositions, all possible anchor modifications.

In some embodiments, for all of the foreign peptides in the seed set,new peptide sequences with all possible anchor residue modifications(e.g., MHC class I or class II) are created resulting in a newheteroclitic base set (Expanded set in FIGS. 1-2) that includes all ofthe modifications. In some embodiments, the heteroclitic base set(Expanded set in FIGS. 1-2) also includes the original seed (or base)set (Seed Peptide Set in FIGS. 1-2). In some embodiments, theheteroclitic base set includes amino acid substitutions or non-naturalamino acid analogs at non-anchor residues. The heteroclitic base set isscored for HLA affinity or other metrics as described herein (anotherround of Peptide Filtering and Scoring as shown in FIGS. 1-2). Thescoring predictions may be further updated for pairs of heterocliticpeptide and HLA allele, eliminating pairs where a heteroclitic peptideis predicted to be displayed by an allele but the seed (or base) peptidefrom which it was derived is not predicted to be displayed by theallele. The scoring predictions may also be filtered to ensure thatpredicted binding cores of the heteroclitic peptide displayed by aparticular HLA allele align exactly in position with the binding coresof the respective seed (or base) set foreign peptide for that HLAallele. In some embodiments the scoring predictions are filtered for anHLA allele to ensure that the heteroclitic peptides considered for thatHLA allele are only modified at anchor positions determined by that HLAallele. In some embodiments, heteroclitic peptides are included inexperimental assays such as MIRA (Klinger et al., 2015) to determinetheir immunogenicity with respect to specific HLA alleles. In someembodiments, the methods of Liu et al. (2020b), can be used toincorporate MIRA data for heteroclitic peptides into a model ofpeptide-HLA immunogenicity. In some embodiments, the immunogenicity ofheteroclitic peptides are experimentally determined and their ability toactivate T cells that also recognize the corresponding seed (or base)peptide of the heteroclitic peptide is performed as is known in the art.In some embodiments, these assays of the immunogenicity andcross-reactivity of heteroclitic peptides are performed when theheteroclitic peptides are displayed by specific HLA alleles. In someembodiments, computational predictions of the immunogenicity of aheteroclitic peptide in the context of display by HLA alleles can usedfor scoring such as the methods of Ogishi et al. (2019).

In some embodiments, the next step involves inputting heteroclitic baseset (also referred to as Expanded set as shown in FIGS. 1-2) to OptiVaxto select a compact set of vaccine peptides that maximizes vaccineperformance (Vaccine Performance Optimization; FIGS. 1-2). Vaccineperformance is the population coverage of a vaccine, or the expectednumber peptide-HLA hits produced by a vaccine, or a function ofpopulation coverage and expected number of peptide-HLA hits desired. Insome embodiments, the vaccine immunogenicity metric is a metric thatdescribes the overall immunogenic properties of a vaccine with two ormore peptides In some embodiments, the methods described herein areincluded for running OptiVax. In some embodiments, population coveragemeans the proportion of a subject population that presents one or moreimmunogenic peptides that activate T cells responsive to a seed foreignpeptide. The metric of population coverage is computed using the HLAhaplotype frequency in a given population such as a representative humanpopulation. In some embodiments, the metric of population coverage iscomputed using marginal HLA frequencies in a population. Maximizingpopulation coverage means selecting a foreign peptide set thatcollectively results in the greatest fraction of the population that hasat least a minimum number (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) ofimmunogenic peptide-HLA bindings based on proportions of HLA haplotypesin a given population (e.g., representative healthy or diseased humanpopulation). In some embodiments, this process includes the OptiVaxselection of heteroclitic peptides (as described in this disclosure)that activate T cells that respond to their corresponding seed (or base)peptide and the heteroclitic base peptides to improve populationcoverage. In some embodiments, the seed (or base) foreign peptides arealways included in the final vaccine design to guard against thepossibility that heteroclitic peptides will not produce immunity thatreacts with the native seed (or base) foreign peptides. In someembodiments, peptides are only considered as candidates for a vaccinedesign if they have been observed to be immunogenic in clinical data oranimal models.

In some embodiments, a candidate vaccine peptide is eliminated fromvaccine inclusion if it activates T cells that recognize self-peptides(e.g., this can be achieved at the first and/or second round of PeptideFiltering and Sorting as shown in FIGS. 1-2). Testing a vaccine peptidefor its ability to activate T cells that recognize self-peptides can beexperimentally accomplished by the vaccination of animal models followedby ELISPOT or other immunogenicity assay or with human tissue protocols.In both cases, models with HLA alleles that present the vaccine peptideare used. In some embodiments, human primary blood mononuclear cells(PBMCs) are stimulated with a vaccine peptide, the T cells are allowedto grow, and then T cell activation with a self-peptide is assayed asdescribed in Tapia-Calle et al. (2019) or other methods as known in theart. In some embodiments, computational predictions of the ability of apeptide to activate T cells that also recognize self-peptides can beutilized. These predictions can be based upon the modeling of theoutward facing residues from the peptide-HLA complex and theirinteractions with other peptide residues. In some embodiments, acandidate vaccine peptide is eliminated from vaccine inclusion orexperimentally tested for cross-reactivity if it is predicted toactivate T cells that also recognize self-peptides based upon thestructural similarity of the peptide-MHC complex of the candidatepeptide and the peptide-MHC complex of a self-peptide. One method forthe prediction of peptide-MHC structure is described by Park et al.(2013).

In some embodiments, a candidate heteroclitic vaccine peptide iseliminated from vaccine inclusion if it does not activate T cells thatrecognize its corresponding seed foreign peptide (second round ofPeptide Filtering and Scoring, FIGS. 1-2). Testing a candidateheteroclitic peptide for its ability to activate T cells that recognizeits corresponding seed (or base) foreign peptide can be experimentallyaccomplished by the vaccination of animal models followed by ELISPOT orother immunogenicity assay or with human tissue protocols. In bothcases, models with HLA alleles that present the heteroclitic peptide areused. In some embodiments, human PBMCs are stimulated with theheteroclitic peptide, the T cells are allowed to grow, and then T cellactivation with the seed (or base) foreign peptide is assayed asdescribed in Tapia-Calle et al. (2019) or using other methods known inthe art. In some embodiments, computational predictions of the abilityof a heteroclitic peptide to activate T cells that also recognize thecorresponding seed (or base) foreign peptide can be utilized. Thesepredictions can be based upon the modeling of the outward facingresidues from the peptide-HLA complex and their interactions with otherpeptide residues. In some embodiments, the structural similarity of thepeptide-MHC complex of a heteroclitic peptide and the peptide-MHCcomplex of the corresponding seed (or base) foreign is used to qualifyheteroclitic peptides for vaccine inclusion or to require experimentalimmunogenicity testing before vaccine inclusion.

FIG. 3 (MHC class I) and FIG. 4 (MHC class II) show the predictedpopulation coverage of OptiVax-Robust selected single target-specificvaccines with differing number of peptides designed for the KRASmutations G12D, G12V, G12R, G12C, and G13D. FIGS. 4-5 show that as thenumber of peptides increases for a vaccine, its predicted populationcoverage increases. The population coverage shown in FIGS. 4-5 are ofthose individuals that have the specific mutation that the vaccine isdesigned to cover. An increase in peptide count will also typicallycause the average number of peptide-HLA hits in each individual toincrease in the population.

OptiVax can be used to design a vaccine to maximize thefraction/proportion of the population whose HLA molecules are predictedto bind to and display at least p peptides from the vaccine. In someembodiments, this prediction includes experimental immunogenicity datato directly predict at least p peptides will be immunogenic. The numberp is input to OptiVax, and OptiVax can be run multiple times withvarying values for p to obtain a predicted optimal foreign peptide setfor different peptide counts p. Larger values of p will increase theredundancy of a vaccine at the cost of more peptides to achieve adesired population coverage. In some embodiments, it may not be possibleto achieve a given population coverage given a specific heterocliticbase set. In some embodiments, the number p is a function of the desiredsize of a vaccine.

The methods described herein can be used to design separate vaccineformulations for MHC class I and class II based immunity.

In some embodiments, this procedure is used to create a vaccine for anindividual. In some embodiments, the foreign peptides present in theindividual are determined by sequencing the individual's tumor RNA orDNA, and identifying mutations that produce foreign peptides. Oneembodiment of this is described in U.S. Ser. No. 10/738,355B2. In someembodiments, peptide sequencing methods are used to identify foreignpeptides in the individual. One embodiment of this is described inUS20110257890A1. In some embodiments, the foreign peptides used for theindividual's vaccine are selected when a foreign peptide or foreignpeptide encoding RNA observed in a specimen from the individual ispresent at a predetermined level. The foreign peptides in the individualare used to construct a vaccine as described in the disclosure herein.For vaccine design OptiVax is provided a diplotype comprising the HLAtype of the individual. In an alternative embodiment, the HLA type of anindividual is separated into multiple diplotypes with frequencies thatsum to one, where each diplotype comprises one or more HLA alleles fromthe individual and a notation that the other allele positions should notbe evaluated. The use of multiple diplotypes will cause OptiVax'sobjective function to increase the chance that immunogenic peptides willbe displayed by all of the constructed diplotypes.

FIG. 10 shows the vaccine performance (predicted number of peptide-HLAhits) of ten example G12V MHC class I vaccines for a single individualwith the MHC class I HLA diplotype HLA-A02:03, HLA-A11:01, HLA-B55:02,HLA-B58:01, HLA-C03:02, HLA-C03:03. OptiVax was used to design ten G12VMHC class I vaccines for this HLA diplotype with peptide counts rangingfrom 1 to 10. For the results in FIG. 10, OptiVax was run with sixsynthetic diplotypes, each equally weighted, each with one HLA allelefrom the individual's HLA diplotype, and the other allele positionsmarked to not be evaluated. The 10 peptide vaccine in FIG. 10 comprisesSEQ ID NO: 3 (GAVGVGKSL), SEQ ID NO: 4 (LMVVGAVGV), SEQ ID NO: 7(VVGAVGVGK), SEQ ID NO: 14 (GPVGVGKSV), SEQ ID NO: 69 (LMVVGAVGI), SEQID NO: 72 (LMVVGAVGL), SEQ ID NO: 131 (GAVGVGKSM), SEQ ID NO: 138(GPVGVGKSA), SEQ ID NO: 142 (VTGAVGVGK), and SEQ ID NO: 198 (VAGAVGVGM).Two peptides, SEQ ID NO: 3 (GAVGVGKSL) and SEQ ID NO: 131 (GAVGVGKSM),are predicted to bind two of the HLA alleles with an affinity of 50 nMor less.

MHC Class I Vaccine Design Procedure

In some embodiments, MHC class I vaccine design procedures consist ofthe following computations steps.

In some embodiments, the inputs for the computation are:

-   -   P_(1 . . . n): Peptide sequence (length n) containing the        neoantigen or pathogenic target(s) of interest (e.g., KRAS G12D,        KRAS G12V, KRAS G12R, KRAS G12C, KRAS G13D). P_(i) denotes the        amino acid at position i.    -   t: Position of target mutation in P, t∈[1, . . . , n] (e.g.,        t=12 for KRAS G12D).    -   τ₁: Threshold for potential presentation of peptides by MHC for        peptide-MHC scoring (e.g., 500 nM binding affinity)    -   τ₂: Threshold for predicted display of peptides by MHC for        peptide-MHC scoring (e.g., 50 nM binding affinity)    -   : Set of HLA alleles (for HLA-A, HLA-B, HLA-C loci)    -   F:        ³→        : Population haplotype frequencies (for OptiVax optimization and        coverage evaluation).    -   N: Parameter for EvalVax and OptiVax objective function.        Specifies minimum number of predicted per-individual hits for        population coverage objective to consider the individual        covered. Default=1 (computes P(n≥1) population coverage).

In some embodiments, Peptide-HLA Scoring Functions used are:

-   -   ScorePotential: P×        →        : Scoring function mapping a (peptide, HLA allele) pair to a        prediction of peptide-HLA display. If predicted affinity ≤τ₁,        then returns 1, else returns 0. Options include MHCflurry,        NetMHCpan, PUFFIN, ensembles, or alternative metrics or software        may be used, including models calibrated against immunogenicity        data.    -   SCOREDISPLAY: P×        →        : Scoring function mapping a (peptide, HLA allele) pair to a        prediction of peptide-HLA display. If predicted affinity ≤τ₂,        then returns 1, else returns 0. Options include MHCflurry,        NetMHCpan, PUFFIN, ensembles, or alternative metrics or software        may be used, including models calibrated against immunogenicity        data.

Next, from the seed protein sequence (P), a set

of windowed native peptides spanning the protein sequence(s) isconstructed. In some embodiments, 9-mers are produced, but this processcan be performed with any desired window lengths and the resultingpeptide sets combined.

={P _(j . . . j+8) |j∈[t−8, . . . ,t],j≠{t−7,t}}

The second condition j≠{t−md 7, t} excludes peptides where the mutationat t is in positions P2 or P9 of the windowed 9-mer peptide (i.e., theanchor positions).

Next, each peptide sequence in

is scored against all HLA alleles in

for potential presentation using SCOREPOTENTIAL (with threshold τ₁=500nM) and store results in a |

|×|

| matrix S:S[p,h]=SCOREPOTENTIAL(p,h)∀p∈

,h∈

-   -   Note that S is a binary matrix where 1 indicates the HLA is        predicted to potentially present the peptide, and 0 indicates no        potential presentation.        Define base set of peptides B⊆        :        B={p∈        |∃h s.t.S[p,h]=1}

Thus, B contains the native peptides that are predicted to bepotentially presented by at least 1 HLA.

Create a Set of all Heteroclitic Peptides B′ Stemming from Peptides inB:

$B^{\prime} = {{\bigcup\limits_{b \in B}{ANCHOR}} - {{MODIFIED}(b)}}$

-   -   where ANCHOR-MODIFIED(b) returns a set of all 399        anchor-modified peptides stemming from b (with all possible        modifications to the amino acids at P2 and P9).

Next, all heteroclitic candidate peptides in B′ are scored against allHLA alleles in

for predicted display using SCOREDISPLAY (with threshold T2=50 nM), andstore results in binary |B′|×|

| matrix S₁′:S ₁′[b′,h]=SCOREDISPLAY(b′,h)∀b′∈B′,h∈

Next, an updated scoring matrix S₂′ is computed for heterocliticpeptides conditioned on the potential presentation of the correspondingbase peptides by each HLA:

${S_{2}^{\prime}\left\lbrack {b^{\prime},h} \right\rbrack} = \left\{ {{\begin{matrix}{{S_{1}^{\prime}\left\lbrack {b^{\prime},h} \right\rbrack},} & {{{if}\mspace{14mu}{S\left\lbrack {b,h} \right\rbrack}} = 1} \\{0,} & {otherwise}\end{matrix}{\forall{b^{\prime} \in B^{\prime}}}},{h \in \mathcal{H}}} \right.$

-   -   where each heteroclitic peptide b′∈B′ is a mutation of base        peptide b∈B. This condition enforces that if h was not predicted        to potentially present b, then all heteroclitic peptides b′        derived from b will not be displayed by h (even if h would        otherwise be predicted to display b′).

In some embodiments, OptiVax-Robust is used to design a final peptideset from the union of base peptides and heteroclitic peptides B∪B′ (withcorresponding scoring matrices S and S₂′ for B and B′, respectively).Let

_(k) denote the compact set of vaccine peptides output by OptiVaxcontaining k peptides. Note that

_(k+1) is not necessarily a superset of

_(k). (In alternate embodiments, OptiVax can be used to augment the baseset B with peptides from B′ using scoring matrix S₂′ to return set

_(k), and the final vaccine set

_(k+|B|) consists of peptides B∪

_(k).)

In some embodiments, this procedure is repeated independently for eachtarget of interest, and the resulting independent vaccine sets can bemerged into a combined vaccine as described below.

MHC Class II Vaccine Design Procedure

In some embodiments, MHC class II vaccine design procedures consist ofthe following computations steps.

In some embodiments, the inputs for the computation are:

-   -   P_(1 . . . n): Peptide sequence(s) (length n) containing the        neoantigen(s) of interest (e.g., KRAS G12D, KRAS G12V, KRAS        G12R, KRAS G12C, KRAS G13D). P_(i) denotes the amino acid at        position i.    -   t: Position of target mutation in P, t∈[1, . . . , n] (e.g.,        t=12 for KRAS G12D).    -   τ₁: Threshold for potential presentation of peptides by MHC for        peptide-MHC scoring (e.g., 500 nM binding affinity)    -   τ₂: Threshold for predicted display of peptides by MHC for        peptide-MHC scoring (e.g., 50 nM binding affinity)    -   : Set of HLA alleles (for HLA-DR, HLA-DQ, HLA-DP loci)    -   F:        ³→        : Population haplotype frequencies (for OptiVax optimization and        coverage evaluation).    -   N: Parameter for EvalVax and OptiVax objective function.        Specifies minimum number of predicted per-individual hits for        population coverage objective to consider the individual        covered. Default=1 (computes P(n≥1) population coverage).

In some embodiments, Peptide-HLA Scoring Functions used are:

-   -   SCOREPOTENTIAL: P×        →        : Scoring function mapping a (peptide, HLA allele) pair to a        prediction of peptide-HLA display. If predicted affinity ≤τ₁,        then returns 1, else returns 0.    -   Options include NetMHCIIpan, PUFFIN, ensembles, or alternative        metrics or software may be used, including models calibrated        against immunogenicity data.    -   SCOREDISPLAY: P×        →        : Scoring function mapping a (peptide, HLA allele) pair to a        prediction of peptide-HLA display. If predicted affinity ≤τ₂,        then returns 1, else returns 0. Options include NetMHCIIpan,        PUFFIN, ensembles, or alternative metrics or software may be        used, including models calibrated against immunogenicity data.    -   FindCore: P×        →[1, . . . , n]: Function mapping a (peptide, HLA allele) pair        to a prediction of the 9-mer binding core. The core may be        specified as the offset position (index) into the peptide where        the core begins.

Next, from the seed protein sequence (P), a set

of peptides spanning the protein sequence are constructed. Here, weextract all windowed peptides of length 13-25 spanning the targetmutation, but this process can be performed using any desired windowlengths (e.g., only 15-mers).

$\mathcal{P} = {\bigcup\limits_{k \in {\lbrack{13,\mspace{14mu}\ldots\mspace{14mu},25}\rbrack}}\mathcal{P}_{k}}$𝒫_(k) = {𝒫_(j  …  j + (k − 1))|j ∈ [t − (k − 1) , …  , t]}

-   -   where        _(k) contains all sliding windows of length k, which are        combined to form        . Note that here (unlike MHC class I), no peptides are excluded        based on binding core or anchor residue positions (for MHC class        II, filtering is performed in Paragraph 0063).

Next, each peptide sequence in P is scored against all HLA alleles in

for potential presentation using SCOREPOTENTIAL (with threshold τ₁=500nM) and store results in a |

|×|

| matrix S₁:S ₁[p,h]=SCOREPOTENTIAL(p,h)∀p∈

,h∈

-   -   Note that S₁ is a binary matrix where 1 indicates the HLA is        predicted to potentially present the peptide, and 0 indicates no        potential presentation.

For each (peptide, HLA allele) pair (p, h), identify/predict the 9-merbinding core using FINDCORE. The predicted binding core is recorded in amatrix C:C[p,h]=FINDCORE(p,h)∀p∈

,h∈

Next, an updated scoring matrix S₂ is computed for native peptides in

:

${S_{2}\left\lbrack {p,h} \right\rbrack} = \left\{ {{\begin{matrix}{{S_{1}\left\lbrack {p,h} \right\rbrack}\ ,} & {{{if}\mspace{14mu}{C\left\lbrack {p,h} \right\rbrack}\ {specifies}\mspace{14mu} P_{t\mspace{14mu}}{at}\mspace{14mu} a\mspace{14mu}{non}} - {{anchor}\mspace{14mu}{position}\mspace{14mu}{inside}\mspace{14mu}{core}}} \\{0,} & {otherwise}\end{matrix}{\forall{p \in \mathcal{P}}}},{h \in \mathcal{H}}} \right.$

-   -   where P_(t) is the target residue of interest (e.g., the        mutation site of KRAS G12D). This condition enforces the target        residue to fall within the binding core at a non-anchor position        for all (peptide, HLA allele) pairs with non-zero scores in S₂,        and allows the binding core to vary by allele per peptide (as        the binding cores of a particular peptide may differ based on        the HLA allele presenting the peptide). Thus, for each pair (p,        h), if the predicted binding core C[p, h] specifies the target        residue P_(t) at an anchor position (P1, P4, P6, or P9 of the        9-mer core), or if P_(t) is not contained within the binding        core, then S₂ [p, h]=0. In an alternate embodiment, P_(t) can be        located outside of the core or inside the core in a non-anchor        position.

Next, OptiVax-Robust is run with peptides

and scoring matrix S₂ to identify a non-redundant base set of peptidesB⊆

. (In alternate embodiments, B can be chosen as the entire set

rather than identifying a non-redundant base set.)

Next, a set of all heteroclitic peptides B′ is created stemming frompeptides in B:

$B^{\prime} = {\bigcup\limits_{b \in {\bigcup B}}\left\{ {\left. {{ANCHOR} - {{{MODIFIED}\left( {b,c} \right)}{\forall c}}} \middle| {\exists{h\mspace{14mu}{s.t.\mspace{14mu}{S_{2}\left\lbrack {b,h} \right\rbrack}}}} \right. = 1} \right\}}$

-   -   where ANCHOR-MODIFIED(b,c) returns a set of all 20⁴−1        anchor-modified peptides stemming from b with all possible        modifications to the amino acids at P1, P4, P6, and P9 of the        9-mer binding core c. Thus, for each base peptide b, the        heteroclitic set B′ contains all anchor-modified peptides b′        with modifications to all unique cores of b identified for any        HLA alleles that potentially present b with a valid core        position as indicated by scoring matrix S₂.

Next, all heteroclitic candidate peptides in B′ are scored against allHLA alleles in

for predicted display using SCOREDISPLAY (with threshold τ₂=50 nM), andstore results in binary |B′|×|

| matrix S₁′:S ₁′[b′,h]=ScoreDisplay(b′,h)∀b′∈B′,h∈

For each (heteroclitic peptide, HLA allele) pair (b′,h),identify/predict the 9-mer binding core using FINDCORE. The predictedbinding core is recorded in a matrix C′:C′[b′,h]=FINDCORE(b′,h)∀b′∈B′,h∈

An updated scoring matrix S₂′ is computed for heteroclitic peptidesconditioned on the identified binding cores of a heteroclitic and basepeptides occurring at the same offset by a particular HLA:

${S_{2}^{\prime}\left\lbrack {b^{\prime},h} \right\rbrack} = \left\{ {{\begin{matrix}{{S_{1}^{\prime}\left\lbrack {b^{\prime},h} \right\rbrack},} & {{{if}\mspace{14mu}{C^{\prime}\left\lbrack {b^{\prime},h} \right\rbrack}} = {C\left\lbrack {b,h} \right\rbrack}} \\{0,} & {otherwise}\end{matrix}{\forall{b^{\prime} \in B^{\prime}}}},{h \in \mathcal{H}}} \right.$

-   -   where each heteroclitic peptide b′∈B′ is a mutation of base        peptide b∈B. This condition enforces the binding core of the        heteroclitic peptide b′ to be at the same relative position as        the base peptide b, and, implicitly, enforces that the target        residue P_(t) still falls in a non-anchor position within the        9-mer binding core (Step 3).

An updated scoring matrix S₃′ is computed for heteroclitic peptidesconditioned on the potential presentation of the corresponding basepeptides by each HLA:

${S_{3}^{\prime}\left\lbrack {b^{\prime},h} \right\rbrack} = \left\{ {{\begin{matrix}{{S_{2}^{\prime}\left\lbrack {b^{\prime},h} \right\rbrack},} & {{{if}\mspace{14mu}{S\left\lbrack {b,h} \right\rbrack}} = 1} \\{{0,}\ } & {otherwise}\end{matrix}{\forall{b^{\prime} \in B^{\prime}}}},{h \in \mathcal{H}}} \right.$

-   -   where each heteroclitic peptide b′∈B′ is a mutation of base        peptide b∈B. This condition enforces that if h was not predicted        to display b, then all heteroclitic peptides b′ derived from b        will not be displayed by h (even if h would otherwise be        predicted to display b′).

OptiVax-Robust is used to design a final peptide set from the union ofbase peptides and heteroclitic peptides B∪B′ (with corresponding scoringmatrices S₂ and S₃′ for B and B′, respectively). Let

_(k) denote the compact set of vaccine peptides output by OptiVaxcontaining k peptides. Note that

_(k+1) is not necessarily a superset of

_(k). (In alternate embodiments, OptiVax can be used to augment the baseset B with peptides from B′ using scoring matrix S₂′ to return set

_(k), and the final vaccine set

_(k+|B|) consists of peptides B∪

_(k).)

In some embodiments, this procedure is repeated independently for eachsingle target of interest, and the resulting independent vaccine setscan be merged into a combined vaccine as described below.

Methods for Combining Multiple Vaccines

The above described methods will produce an optimized foreign peptideset for one or more targets. In some embodiments, a method is providedfor designing separate vaccines for MHC class I and class II basedimmunity for multiple targets (e.g., two or more targets such as KARSG12D and KRAS G12V).

In some embodiments, a method is disclosed for producing a combinedpeptide vaccine for multiple targets by using a table of presentationsfor a disease that is based upon empirical data from sources such as theCancer Genome Atlas (TCGA). FIG. 5 shows one embodiment for factoringdisease presentation type probabilities (pancreatic cancer, colon/rectumcancer, and bronchus/lung cancer) by probability, for each diseasepresentation, of target presented for various KRAS mutation targets(KRAS G12D, KRAS G12V, and KRAS G12R). A presentation is a unique set oftargets that are presented by one form of a disease (e.g., distinct typeof cancer as shown in FIG. 5). For each presentation, FIG. 5 shows anexample of the probability of that presentation, and the probabilitythat a given target is observed. For a given presentation, there can beone or more targets, each having a probability. In some embodiments, themethod for multi-target vaccine design will allocate peptide resourcesfor inducing disease immunity based on the presentation and respectivetarget probabilities as shown in FIG. 5, for example. In someembodiments, presentations correspond to the prevalence of targets indifferent human populations or different risk groups. The probability ofa target in a population is computed by summing for each possiblepresentation the probability of that presentation times the probabilityof the target in that presentation.

Referring to FIG. 6, in some embodiments, the method first includesdesigning an individual peptide vaccine for each target to create acombined vaccine design for multiple targets. This initially results insets of target-specific vaccine designs. In some embodiments, themarginal vaccine performance of each target-specific vaccine at size kis defined by vaccine performance at size k minus the vaccineperformance of the vaccine at size k minus one (see FIGS. 3-4). Thecomposition of a vaccine may change as the number of peptides used inthe vaccine increases, and thus for computing contributions to acombined vaccine the marginal vaccine performance of eachtarget-specific vaccine is used instead of a specific set of peptides.

In some embodiments, the weighted marginal vaccine performance of atarget-specific vaccine design for each target specific vaccine size iscomputed as shown in FIG. 6. For a given target specific vaccine size,its weighted vaccine performance is computed by multiplying its vaccineperformance times the probability of the target in the population (e.g.,by using values as shown in FIG. 5). The marginal weighted vaccineperformance for a target specific vaccine is its weighted coverage atsize k minus its coverage a size k minus one (e.g., see FIGS. 3-4). Themarginal weighted vaccine performance of a target specific vaccine ofsize one is its weighted vaccine performance. The marginal weightedvaccine performances for all vaccines are combined into a single list,and the combined list is sorted from largest to least by the weightedmarginal vaccine performances of the target specific vaccines as shownin FIG. 6. The combined vaccine of size n is then determined by thefirst n elements of this list. The peptides for the combined vaccine aredetermined by the individual peptide target vaccines whose sizes add ton and whose weighted vaccine performances sums to the same sum as thefirst n elements of the sorted list. This maximizes the vaccineperformance of the combined vaccine of size n.

In some embodiments, the combined multiple target vaccine can bedesigned on its overall predicted coverage for the disease describeddepending on the presentation table used (e.g., see FIG. 5), by itspredicted coverage for a specific indication, and/or by its predictedcoverage for a specific target by adjusting the weighting used forvaccine performance accordingly. Once a desired level of coverage isselected, the peptides of the combined vaccine are determined by thecontributions of target-specific designs. For example, if the combinedvaccine includes a target-specific vaccine of size k, then the vaccinepeptides for this target at size k are used in the combined vaccine.

As an example of one embodiment, FIG. 5 shows three mutations (KRASG12D, G12V, and G12R) and their respective probabilities of occurring inan individual with pancreatic cancer. FIG. 3 (MHC class I) and FIG. 4(MHC class II) show the population coverage of target-specific vaccinesfor the KRAS G12D, G12V, G12R, G12C, and G13D targets using the methodsfor vaccines described herein. The marginal population coverage of eachtarget-specific vaccine at a given vaccine size is the improvement incoverage at that size and the size less one. The coverage with nopeptides is zero. The marginal coverage of each target-specific vaccineis multiplied by the probability of the target in the population asdetermined by the proportions as shown in FIG. 5 for the pancreas(pancreatic cancer). These weighted marginal coverages of alltarget-specific vaccines are sorted to determine the besttarget-specific compositions, and the resulting list describes thecomposition of a combined vaccine at each size k by taking the first kelements of the list. As an example of one embodiment, FIG. 7 (MHC ClassI) and FIG. 8 (MHC Class II) show the target specific contributions ateach vaccine size for a combined KRAS vaccine for the three mutationsKRAS G12D, G12V, and G12R. The methods for combined vaccine protocoldescribed herein was used to compute the examples in FIGS. 7 and 8. Ateach combined vaccine size, different components of the target-specificvaccines are utilized. Table 1 (below) contains the peptides present inindependent (single target) and combined (multiple target) MHC class Ivaccine designs for the KRAS G12D, G12V, G12R, G12C, and G13D targets.Table 2 (below) contains the contains the peptides present inindependent (single target) MHC class II vaccine designs for the KRASG12D, G12V, G12R, G12C, and G13D targets, and any subset of theindividual/single target vaccines can be combined to create an MHC classII vaccine for two or more multiple targets. For alternate embodiments,Sequence Listing provides heteroclitic peptides useful in MHC class Ivaccines for the KRAS G12D, G12V, G12R, G12C, and G13D targets.

Combined Vaccine Design Procedure

In some embodiments, the procedure described herein is used to combineindividual compact vaccines optimized for different targets into asingle optimized combined vaccine.

In some embodiments, the computational inputs for the procedure are:

-   -   : Set of neoantigen or pathogenic targets of interest (e.g.,        KRAS G12D, KRAS G12V, KRAS G12R)    -   : Vaccine sets optimized individually for each target. Let        _(t,k) denote the optimal vaccine set of exactly k peptides for        target t∈        (e.g., as computed by the procedures describe above). Note that        _(t,k+1) may not necessarily be a superset of        _(t,k).    -   W:        →[0,1]: Target weighting function mapping each target t∈        to a probability or weight of tin a particular presentation of        interest (e.g., pancreatic cancer; see Exhibit A, Table 1 for        example).    -   POPULATIONCOVERAGE:        →[0,1]: Function mapping a peptide set into population coverage        (e.g., EvalVax). This function may also take as input additional        parameters, including HLA haplotype frequencies and a minimum        per-individual number of peptide-HLA hits N (here, we compute        coverage as P(n≥1) using EvalVax-Robust).

For each target t (individually) and vaccine size (peptide count) k, theunweighted population coverage c_(t,k) is computed:c _(t,k)=PopulationCoverage(

_(t,k))∀t∈

k

-   -   Note that for each target t, c_(t,k) is generally monotonically        increasing and concave down for increasing values of k (each        additional peptide increases coverage but with decreasing        returns).

For each target t (individually), the marginal coverage m_(t,k) iscomputed of the k-th peptide added to the vaccine set:

$m_{t,k} = \left\{ {{\begin{matrix}c_{t,k} & {{{if}\mspace{14mu} k} = 1} \\{{c_{t,k} - c_{t,{k - 1}}},} & {otherwise}\end{matrix}{\forall{t \in \mathcal{T}}}},k} \right.$

-   -   Note that for each target t, m_(t,k) should be a monotonically        decreasing function in k (by Step 1 above).

The weighted marginal population coverage {tilde over (m)}_(t,k) iscomputed using weights of each target in W:{tilde over (m)} _(t,k) =W(t)·m _(t,k) ∀t∈

,k

-   -   The weighted marginal population coverage gives the effective        marginal coverage of the k-th peptide in the vaccine weighted by        the prevalence of the target in the presentation (by        multiplication with the probability/weight of the target in the        presentation).

The individual vaccines are combined into a combined vaccine via theMERGEMULTI procedure called on the weighted marginal population coveragelists {tilde over (m)}_(t)=[{tilde over (m)}_(t,k), k∈1, 2, . . . ].FIG. 9 shows an example Python implementation of the MERGEMULTIfunction. This procedure takes as input multiple sorted (descending)lists and merges them into a single sorted (descending) list. Let Mindicate the output of MERGEMULTI where each element M_(k) contains boththe marginal weighted coverage and source (target) of the k-th peptidein the combined vaccine. The combined vaccine contains peptides fromdifferent targets. In particular, the combined vaccine with k peptidescontains C_(t,k)=Σ_(j≤k)]]{M_(k) from t} peptides from target t. Notethat C_(t,k)∈[0, . . . , k] and Σ_(t) C_(t,k)=k (C_(t,k) gives thedistribution of the k peptides in the combined vaccine across thetargets).

The optimal combined vaccine set

_(k) is defined as:

${\hat{v}}_{k} = {\bigcup\limits_{t \in \mathcal{T}}v_{t,C_{t,k}}}$

Thus, the combined vaccine with k peptides is the combination of theoptimal individual (C_(t,k))-peptide vaccines. The marginal weightedcoverage values of the combine vaccine M_(k) can be cumulatively summedover k to give the overall effective (target-weighted) populationcoverage of the combined vaccine containing k peptides as Σ_(j≤K)M_(k)(taking into account both the probabilities/weights of the targets inthe presentation and the expected population coverage of peptides basedon HLA display). The final vaccine size k can vary based upon thespecific population coverage goals of the vaccine.

MHC Class I Peptide Sequences

In some embodiments, a peptide vaccine (single target or combinedmultiple target vaccine) comprises about five, ten, or twenty MHC classI peptides with each peptide consisting of 8 or more amino acids. Insome embodiments, an MHC class I peptide vaccine is intended for one ormore of the KRAS G12D, G12V, and G12R targets. In some embodiments, theamino acid sequence of a first peptide in a five-peptide combinedvaccine comprises SEQ ID NO: 1.

(SEQ ID NO: 1) GADGVGKSM.In some embodiments, the amino acid sequence of a second peptide in afive-peptide combined vaccine comprises SEQ ID NO: 2.

(SEQ ID NO: 2) LMVVGADGV.In some embodiments, the amino acid sequence of a third peptide in afive-peptide combined vaccine comprises SEQ ID NO: 3.

(SEQ ID NO: 3) GAVGVGKSL.In some embodiments, the amino acid sequence of a fourth peptide in afive-peptide combined vaccine comprises SEQ ID NO: 4.

(SEQ ID NO: 4) LMVVGAVGV.In some embodiments, the amino acid sequence of a fifth peptide in afive-peptide combined vaccine comprises SEQ ID NO: 5.

(SEQ ID NO: 5) VTGARGVGK.An example combined vaccine for the KRAS G12D, G12V, and G12R targetswith five peptides (SEQ ID NO: 1 to SEQ ID NO: 5) is predicted to have aweighted population coverage of 0.3620.

In some embodiments, any one of the peptides (peptides 1-5) in thefive-peptide vaccine comprise an amino acid sequence 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identicalto SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:5.

In some embodiments, the amino acid sequence of peptides 1 to 5 in aten-peptide combined vaccine comprise SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. In some embodiments, the aminoacid sequence of a sixth peptide in a ten-peptide combined vaccinecomprises SEQ ID NO: 6.

(SEQ ID NO: 6) VMGAVGVGK.In some embodiments, the amino acid sequence of a seventh peptide in aten-peptide combined vaccine comprises SEQ ID NO: 7.

(SEQ ID NO: 7) VVGAVGVGK.In some embodiments, the amino acid sequence of an eight peptide in aten-peptide combined vaccine comprises SEQ ID NO: 8.

(SEQ ID NO: 8) GARGVGKSY.In some embodiments, the amino acid sequence of a ninth peptide in aten-peptide combined vaccine comprises SEQ ID NO: 9.

(SEQ ID NO: 9) GPRGVGKSA.In some embodiments, the amino acid sequence of a tenth peptide in aten-peptide combined vaccine comprises SEQ ID NO: 10.

(SEQ ID NO: 10) LMVVGARGV.An example combined vaccine for the KRAS G12D, G12V, and G12R targetswith ten peptides (SEQ ID NO: 1 to SEQ ID NO: 10) is predicted to have aweighted population coverage of 0.4374.

In some embodiments, any one of the peptides (peptides 1-10) in theten-peptide vaccine comprise an amino acid sequence 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identicalto SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:10.

In some embodiments, the amino acid sequence of peptides 1 to 10 in atwenty-peptide combined vaccine comprise SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. In some embodiments, the aminoacid sequence of an 11^(th) peptide in a twenty-peptide combined vaccinecomprises SEQ ID NO: 11.

(SEQ ID NO: 11) GADGVGKSL.In some embodiments, the amino acid sequence of a 12^(th) peptide in atwenty-peptide combined vaccine comprises SEQ ID NO: 12.

(SEQ ID NO: 12) GADGVGKSY.In some embodiments, the amino acid sequence of a 13^(th) peptide in atwenty-peptide combined vaccine comprises SEQ ID NO: 13.

(SEQ ID NO: 13) GYDGVGKSM.In some embodiments, the amino acid sequence of a 14^(th) peptide in atwenty-peptide combined vaccine comprises SEQ ID NO: 14.

(SEQ ID NO: 14) GPVGVGKSV.In some embodiments, the amino acid sequence of a 15^(th) peptide in atwenty-peptide combined vaccine comprises SEQ ID NO: 15.

(SEQ ID NO: 15) LTVVGAVGV.In some embodiments, the amino acid sequence of a 16^(th) peptide in atwenty-peptide combined vaccine comprises SEQ ID NO: 16.

(SEQ ID NO: 16) VVGAVGVGR.In some embodiments, the amino acid sequence of a 17^(th) peptide in atwenty-peptide combined vaccine comprises SEQ ID NO: 17.

(SEQ ID NO: 17) GARGVGKSM.In some embodiments, the amino acid sequence of an 18^(th) peptide in atwenty-peptide combined vaccine comprises SEQ ID NO: 18.

(SEQ ID NO: 18) GPRGVGKSV.In some embodiments, the amino acid sequence of a 19^(th) peptide in atwenty-peptide combined vaccine comprises SEQ ID NO: 19.

(SEQ ID NO: 19) LLVVGARGV.In some embodiments, the amino acid sequence of a 20^(th) peptide in atwenty-peptide combined vaccine comprises SEQ ID NO: 20.

(SEQ ID NO: 20) VAGARGVGM.An example combined vaccine for the KRAS G12D, G12V, and G12R targetswith twenty peptides (SEQ ID NO: 1 to SEQ ID NO: 20) is predicted tohave a weighted population coverage of 0.4604.

In some embodiments, any one of the peptides (peptides 1-20) in thetwenty-peptide vaccine comprise an amino acid sequence 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, andSEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, or SEQ ID NO: 20

Table 1 shows MHC class I peptide sequences described herein includingthe respective SEQ ID NO, amino acid sequence corresponding to the SEQID NO, KRAS protein target (with specific mutation), the seed amino acidsequence (i.e., the amino acid sequence of the wild type KRAS fragment),the amino acid substitution (if any) for heteroclitic peptides atpositions 2 and 9, and notes detailing embodiments in which the peptidemay be included in a 5, 10, or 20 combined peptide vaccine as describedherein. Table 1 also includes additional peptide sequences comprisingSEQ ID NOs: 21-41. In some embodiments, any combination of peptideslisted in Table 1 (SEQ ID NOs: 1-41) may be used to create a combinedpeptide vaccine having between about 2 and about 40 peptides. In someembodiments, any one of the peptides (peptides 1-41; SEQ ID NOs: 1-41)in the combined vaccine comprises an amino acid sequence 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%identical to any of SEQ ID NOs: 1-41.

TABLE 1 Example KRAS Vaccine Peptides (MHC class I) SEQ ID Sequencecorresponding Heteroclitic Heteroclitic NO to SEQ ID Target SeedModification P2 Modification P9 Note SEQ ID GADGVGKSM KRAS GADGVGKSA —A9M Individual KRAS NO: 1 G12D G12D (MHCfluny); Combined (5 peptide)(MHCfluny); Combined (10 peptide) (MHCfluny); Combined (20 peptide)(MHCfluny) SEQ ID LMVVGADGV KRAS LVVVGADGV V2M — Individual KRAS NO: 2G12D G12D (MHCfluny); Individual KRAS G12D (NetMHCpan); Combined (5peptide) (MHCfluny); Combined (10 peptide) (MHCfluny); Combined (20peptide) (MHCfluny) SEQ ID GAVGVGKSL KRAS GAVGVGKSA — A9L IndividualKRAS NO: 3 G12V G12V (MHCfluny); Combined (5 peptide) (MHCfluny);Combined (10 peptide) (MHCfluny); Combined (20 peptide) (MHCfluny) SEQID LMVVGAVGV KRAS LVVVGAVGV V2M — Individual KRAS NO: 4 G12V G12V(MHCfluny); Individual KRAS G12V (NetMHCpan); Combined (5 peptide)(MHCfluny); Combined (10 peptide) (MHCfluny); Combined (20 peptide)(MHCfluny) SEQ ID VTGARGVGK KRAS VVGARGVGK V2T — Individual KRAS NO: 5G12R G12R (MHCfluny); Combined (5 peptide) (MHCfluny); Combined (10peptide) (MHCfluny); Combined (20 peptide) (MHCfluny) SEQ ID VMGAVGVGKKRAS VVGAVGVGK V2M — Individual KRAS NO: 6 G12V G12V (MHCfluny);Combined (10 peptide) (MHCfluny); Combined (20 peptide) (MHCfluny) SEQID VVGAVGVGK KRAS VVGAVGVGK — — Individual KRAS NO: 7 G12V G12V(NetMHCpan); Combined (10 peptide) (MHCfluny); Combined (20 peptide)(MHCfluny) SEQ ID GARGVGKSY KRAS GARGVGKSA — A9Y Individual KRAS NO: 8G12R G12R (MHCfluny); Combined (10 peptide) (MHCfluny); Combined (20peptide) (MHCfluny) SEQ ID GPRGVGKSA KRAS GARGVGKSA A2P — IndividualKRAS NO: 9 G12R G12R (MHCfluny); Combined (10 peptide) (MHCfluny);Combined (20 peptide) (MHCfluny) SEQ ID LMVVGARGV KRAS LVVVGARGV V2M —Individual KRAS NO: 10 G12R G12R (MHCfluny); Individual KRAS G12R(NetMHCpan); Combined (10 peptide) (MHCfluny); Combined (20 peptide)(MHCfluny) SEQ ID GADGVGKSL KRAS GADGVGKSA — A9L Individual KRAS NO: 11G12D G12D (MHCfluny); Combined (20 peptide) (MHCfluny) SEQ ID GADGVGKSYKRAS GADGVGKSA — A9Y Individual KRAS NO: 12 G12D G12D (MHCfluny);Combined (20 peptide) (MHCfluny) SEQ ID GYDGVGKSM KRAS GADGVGKSA A2Y A9MIndividual KRAS NO: 13 G12D G12D (MHCfluny); Combined (20 peptide)(MHCfluny) SEQ ID GPVGVGKSV KRAS GAVGVGKSA A2P A9V Combined (20 NO: 14G12V peptide) (MHCfluny) SEQ ID LTVVGAVGV KRAS LVVVGAVGV V2T —Individual KRAS NO: 15 G12V G12V (NetMHCpan); Combined (20 peptide)(MHCfluny) SEQ ID VVGAVGVGR KRAS VVGAVGVGK — K9R Individual KRAS NO: 16G12V G12V (MHCfluny); Individual KRAS G12V (NetMHCpan); Combined (20peptide) (MHCfluny) SEQ ID GARGVGKSM KRAS GARGVGKSA — A9M Combined (20NO: 17 G12R peptide) (MHCfluny) SEQ ID GPRGVGKSV KRAS GARGVGKSA A2P A9VCombined (20 NO: 18 G12R peptide) (MHCfluny) SEQ ID LLVVGARGV KRASLVVVGARGV V2L — Individual KRAS NO: 19 G12R G12R (NetMHCpan); Combined(20 peptide) (MHCfluny) SEQ ID VAGARGVGM KRAS VVGARGVGK V2A K9MIndividual KRAS NO: 20 G12R G12R (MHCfluny); Combined (20 peptide)(MHCfluny) SEQ ID LTVVGADGV KRAS LVVVGADGV V2T — Individual KRAS NO: 21G12D G12D (NetMHCpan) SEQ ID LLVVGADGV KRAS LVVVGADGV V2L — IndividualKRAS NO: 22 G12D G12D (NetMHCpan) SEQ ID LMVVGADGL KRAS LVVVGADGV V2MV9L Individual KRAS NO: 23 G12D G12D (NetMHCpan) SEQ ID VMGAVGVGR KRASVVGAVGVGK V2M K9R Individual KRAS NO: 24 G12V G12V (NetMHCpan) SEQ IDVMGARGVGK KRAS VVGARGVGK V2M — Individual KRAS NO: 25 G12R G12R(NetMHCpan) SEQ ID GACGVGKSL KRAS GACGVGKSA — A9L Individual KRAS NO: 26G12C G12C (MHCfluny) SEQ ID LMVVGACGV KRAS LVVVGACGV V2M — IndividualKRAS NO: 27 G12C G12C (MHCfluny); Individual KRAS G12C (NetMHCpan) SEQID LTVVGACGV KRAS LVVVGACGV V2T — Individual KRAS NO: 28 G12C G12C(MHCfluny); Individual KRAS G12C (NetMHCpan) SEQ ID VTGACGVGK KRASVVGACGVGK V2T — Individual KRAS NO: 29 G12C G12C (MHCfluny) SEQ IDVVGACGVGR KRAS VVGACGVGK — K9R Individual KRAS NO: 30 G12C G12C(MHCfluny) SEQ ID AADVGKSAM KRAS AGDVGKSAL G2A L9M Individual KRAS NO:31 G13D G13D (MHCfluny); Individual KRAS G13D (NetMHCpan) SEQ IDAEDVGKSAM KRAS AGDVGKSAL G2E L9M Individual KRAS NO: 32 G13D G13D(MHCfluriy) SEQ ID AYDVGKSAM KRAS AGDVGKSAL G2Y L9M Individual KRAS NO:33 G13D G13D (MHCfluriy) SEQ ID DAGKSALTV KRAS DVGKSALTI V2A I9VIndividual KRAS NO: 34 G13D G13D (MHCfluny) SEQ ID GAGDVGKSM KRASGAGDVGKSA — A9M Individual KRAS NO: 35 G13D G13D (MHCfluriy) SEQ IDLQVVGACGV KRAS LVVVGACGV V2Q — Individual KRAS NO: 36 G12C G12C(NetMHCpan) SEQ ID VMGACGVGK KRAS VVGACGVGK V2M — Individual KRAS NO: 37G12C G12C (NetMHCpan) SEQ ID VMGACGVGR KRAS VVGACGVGK V2M K9R IndividualKRAS NO: 38 G12C G12C (NetMHCpan) SEQ ID AADVGKSAL KRAS AGDVGKSAL G2A —Individual KRAS NO: 39 G13D G13D (NetMHCpan) SEQ ID ASDVGKSAL KRASAGDVGKSAL G25 — Individual KRAS NO: 40 G13D G13D (NetMHCpan) SEQ IDASDVGKSAM KRAS AGDVGKSAL G25 L9M Individual KRAS NO: 41 G13D G13D(NetMHCpan)

Additional amino acid sequences of MHC class I heteroclitic peptides areprovided in Sequence Listings (SEQ ID NOs: 67-1522). In someembodiments, any combination of MHC class I peptides disclosed herein(SEQ ID NOs: 1-41 and 67-1522) may be used to create a combined peptidevaccine having between about 2 and about 40 peptides. In someembodiments, any one of the peptides (SEQ ID NOs: 1-41 and 67-1522) inthe combined vaccine comprises or contains an amino acid sequence 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98or 99% identical to any of SEQ ID NOs: 1-41 or 67-1522.

MHC Class II Peptide Sequences

In some embodiments, a peptide vaccine (single target or combinedmultiple target vaccine) comprises about 2 to 40 MHC class II peptideswith each peptide consisting of about 20 amino acids. In someembodiments, an MHC class II peptide vaccine is intended for one or moreof the KRAS G12D, G12V, G12R, G12C, and G13D targets.

Table 2 summarizes MHC class II peptide sequences described hereinincluding the respective SEQ ID NO, amino acid sequence corresponding tothe SEQ ID NO, the amino acid sequence corresponding to the peptide'sbinding core, the KRAS protein target (with specific mutation), the seedamino acid sequence (i.e., the amino acid sequence of the wild type KRASfragment), the seed amino acid sequence of the binding core, and theamino acid substitution (if any) for heteroclitic peptides at positions1, 4, 6, and 9. Table 2 includes peptide sequences comprising SEQ IDNOs: 42-66. SEQ ID NOs: 42-65 (Table 2) encode for recombinant peptides.In some embodiments, any combination of peptides listed in Table 2 (SEQID NOs: 42-66) may be used to create a single target (individual) orcombined peptide vaccine having between about 2 and about 40 peptides.In some embodiments, any one of the peptides (peptides 42-66; SEQ IDNOs: 42-66) in the combined vaccine comprises an amino acid sequence 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98or 99% identical to any of SEQ ID NOs: 42-66.

TABLE 2 Example KRAS Vaccine Peptides (MHC class II) Hetero- Hetero-Hetero- Hetero- clitic clitic clitic clitic Sequence Modifi- Modifi-Modifi- Modifi- SEQ corresponding to cation cation cation cation ID NOSEQ ID Core Target Seed Seed Core P1 P4 P6 P9 Note SEQ EYKFVVFGSDGAGFVVFG KRAS EYKLVVVGADGVG LVVVGADGV L1F V4F A65 V9A Individual ID KS SDGAG12D KS KRAS NO: G12D 42 (NetMHCIIpan) SEQ EYKFVVIGNDGAG FVVIG KRASEYKLVVVGADGVG LVVVGADGV L1F V4I A6N V9A Individual ID KSALTIQLIQN NDGAG12D KSALTIQLIQN KRAS NO: G12D 43 (NetMHCIIpan) SEQ EYKFVVLGADGAG FVVLGKRAS EYKLVVVGADGVG LVVVGADGV L1F V4L — V9A Individual ID KS ADGA G12D KSKRAS NO: G12D 44 (NetMHCIIpan) SEQ MTEYKFVVSGADG FVVSG KRASMTEYKLVVVGADG LVVVGADGV L1F V4S — V9I Individual ID IGKSALT ADGI G12DVGKSALT KRAS NO: G12D 45 (NetMHCIIpan) SEQ MTEYKFVVYGSDG FVVYG KRASMTEYKLVVVGADG LVVVGADGV L1F V4Y A6S V9I Individual ID IGKSALT SDGI G12DVGKSALT KRAS NO: G12D 46 (NetMHCIIpan) SEQ EYKFVVIGRVGHG FVVIG KRASEYKLVVVGAVGVG LVVVGAVGV L1F V4I A6R V9H Individual ID KS RVGH G12V KSKRAS NO: G12V 47 (NetMHCIIpan) SEQ EYKFVVLGTVGHG FVVLG KRASEYKLVVVGAVGVG LVVVGAVGV L1F V4L A6T V9H Individual ID KS TVGH G12V KSKRAS NO: G12V 48 (NetMHCIIpan) SEQ EYKFVVYGNVGM FVVYG KRAS EYKLVVVGAVGVGLVVVGAVGV L1F V4Y A6N V9M Individual ID GKS NVGM G12V KS KRAS NO: G12V49 (NetMHCIIpan) SEQ EYKIVVAGNVGIGK IVVAG KRAS EYKLVVVGAVGVG LVVVGAVGVL1I V4A A6N V9I Individual ID S NVGI G12V KS KRAS NO: G12V 50(NetMHCIIpan) SEQ TEYKIVVMGNVGY IVVMG KRAS TEYKLVVVGAVGV LVVVGAVGV L1IV4M A6N V9Y Individual ID GK NVGY G12V GK KRAS NO: G12V 51 (NetMHCIIpan)SEQ MTEYKFVVFGSRG FVVFG KRAS MTEYKLVVVGARG LVVVGARGV L1F V4F A65 —Individual ID VGKSALT SRGV G12R VGKSALT KRAS NO: G12R 52 (NetMHCIIpan)SEQ MTEYKFVVIGNRG FVVIG KRAS MTEYKLVVVGARG LVVVGARGV L1F V4I A6N —Individual ID VGKSALT NRGV G12R VGKSALT KRAS NO: G12R 53 (NetMHCIIpan)SEQ MTEYKFVVIGVRG FVVIG KRAS MTEYKLVVVGARG LVVVGARGV L1F V4I A6V V9DIndividual ID DGKSALT VRGD G12R VGKSALT KRAS NO: G12R 54 (NetMHCIIpan)SEQ MTEYKFVVMGSRG FVVM KRAS MTEYKLVVVGARG LVVVGARGV L1F V4M A6S V9AIndividual ID AGKSALT GSRGA G12R VGKSALT KRAS NO: G12R 55 (NetMHCIIpan)SEQ VVVIARGVPKSLLT IARGV KRAS VVVGARGVGKSAL GARGVGKSA G1I — G6P A9LIndividual ID I PKSL G12R TI KRAS NO: G12R 56 (NetMHCIIpan) SEQEYKFVVFGNCGAG FVVFG KRAS EYKLVVVGACGVG LVVVGACGV L1F V4F A6N V9AIndividual ID KS NCGA G12C KS KRAS NO: G12C 57 (NetMHCIIpan) SEQEYKFVVSGACGVG FVVSG KRAS EYKLVVVGACGVG LVVVGACGV L1F V4S — — IndividualID KS ACGV G12C KS KRAS NO: G12C 58 (NetMHCIIpan) SEQ EYKFVVSGNCGLGFVVSG KRAS EYKLVVVGACGVG LVVVGACGV L1F V4S A6N V9L Individual ID KS NCGLG12C KS KRAS NO: G12C 59 (NetMHCIIpan) SEQ EYKLVVMGPCGAG LVVM KRASEYKLVVVGACGVG LVVVGACGV — V4M A6P V9A Individual ID KS GPCGA G12C KSKRAS NO: G12C 60 (NetMHCIIpan) SEQ KLVIVGICKVGHSA IVGICK KRASKLVVVGACGVGKS VVGACGVGK V1I A4I G6K K9H Individual ID L VGH G12C AL KRASNO: G12C 61 (NetMHCIIpan) SEQ EYKFVVFGNGDLG FVVFG KRAS EYKLVVVGAGDVGLVVVGAGDV L1F V4F A6N V9L Individual ID KS NGDL G13D KS KRAS NO: G13D 62(NetMHCIIpan) SEQ EYKFVVMGNGDSG FVVM KRAS EYKLVVVGAGDVG LVVVGAGDV L1FV4M A6N V9S Individual ID KS GNGDS G13D KS KRAS NO: G13D 63(NetMHCIIpan) SEQ EYKFVVSGSGDVG FVVSG KRAS EYKLVVVGAGDVG LVVVGAGDV L1FV4S A6S — Individual ID KS SGDV G13D KS KRAS NO: G13D 64 (NetMHCIIpan)SEQ EYKIVVMGRGDMG IVVMG KRAS EYKLVVVGAGDVG LVVVGAGDV L1I V4M A6R V9MIndividual ID KS RGDM G13D KS KRAS NO: G13D 65 (NetMHCIIpan) SEQYKLVVVGAGDVG — KRAS — — — — — — Individual ID KSA G13D KRAS NO: KRASG13D 66 (NetMHCIIpan)

In some embodiments, any combination of MHC class I and/or MHC class IIpeptides disclosed herein (SEQ ID NOs: 1-1522) may be used to create asingle target (individual) or combined peptide vaccine having betweenabout 2 and about 40 peptides. In some embodiments, any one of thepeptides (peptides 1-1522; SEQ ID NOs: 1-1522) in the combined vaccinecomprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to any of SEQ IDNOs: 1-1522.

mRNA and DNA Vaccines

In some embodiments, vaccine peptides are encoded as mRNA or DNAmolecules and are administered for expression in vivo as is known in theart. One example of the delivery of vaccines by mRNA is found in Kranzet al. (2016), incorporated herein by reference. In one embodiment, aconstruct comprises 10 peptides, including a five-peptide MHC class Icombined pancreatic cancer vaccine (targets: KRAS G12D, G12V, G12R) anda five-peptide MHC class II combined pancreatic cancer vaccine (targets:KRAS G12D, G12V, G12R), as optimized by the procedure described herein.Peptides are prepended with a secretion signal sequence at theN-terminus and followed by an MHC class I trafficking signal (MITD)(Kreiter et al., 2008; Sahin et al., 2017). The MITD has been shown toroute antigens to pathways for HLA class I and class II presentation(Kreiter et al., 2008). Here we combine all peptides of each MHC classinto a single construct using non-immunogenic glycine/serine linkersfrom Sahin et al. (2017), though it is also plausible to constructindividual constructs containing single peptides with the same secretionand MITD signals as demonstrated by Kreiter et al. (2008).

In some embodiments, the amino acid sequence encoded by the mRNA vaccinecomprises SEQ ID NO: 1523. Underlined amino acids correspond to thesignal peptide (or leader) sequence. Bolded amino acids correspond toMHC class I (9 amino acids in length; 5 peptides) and MHC class II(13-25 amino acids in length; 5 peptides) peptide sequences. Italicizedamino acids correspond to the trafficking signal.

(SEQ ID NO: 1523) MRVTAPRTLILLLSGALALTETWAGSGGSGGGGSGGGADGVGKSMGGSGGGGSGGLMVVGADGVGGSGGGGSGGGAVGVGKSLGGSGGGGSGGLMVVGAVGVGGSGGGGSGGVTGARGVGKGGSGGGGSGGEYKFVVLGTVGHGKSGGSGGGGSGGEYKIVVAGNVGIGKSGGSGGGGSGGEYKFVVFGSDGAGKSGGSGGGGSGGMTEYKFVVSGADGIGKSALTGGSGGGGSGGMTEYKFVVIGNRGVGKSALTGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQA ASSDSAQGSDVSLTA.

In some embodiments, the vaccine is an mRNA vaccine comprising a nucleicacids sequence encoding the amino acid sequence consisting of SEQ ID NO:1523. In some embodiments, the nucleic acid sequence of the mRNA vaccineencodes for an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO:1523.

In some embodiments, the vaccine is a DNA vaccine comprising a nucleicacids sequence encoding the amino acid sequence consisting of SEQ ID NO:1523. In some embodiments, the nucleic acid sequence of the DNA vaccineencodes for an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO:1523.

In some embodiments, one or more MHC class I and/or MHC class IIpeptides disclosed herein (SEQ ID NO: 1-1522) can be encoded in one ormore mRNA or DNA molecules and administered for expression in vivo. Insome embodiments between about 2 and about 40 peptide sequences areencoded in one or more mRNA constructs. In some embodiments, betweenabout 2 and about 40 peptide sequences are encoded in one or more DNAconstructs (i.e., nucleic acids encoding the amino acids sequencescomprising on or more of SEQ ID NOs: 1-1522). In some embodiments, theamino acid sequence of the mRNA vaccine or the nucleic acid sequence ofthe DNA vaccine encodes for an amino acid sequence 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identicalto any of SEQ ID NOs: 1-1522.

Non-Limiting Embodiments of the Subject Matter

In one aspect, the invention provides for a nucleic acid sequenceencoding two or more amino acid sequences selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.

In some embodiments, the nucleic acid sequence is an immunogeniccomposition. In some embodiments, the nucleic acid sequence isadministered in a construct for expression in vivo. In some embodiments,the in vivo administration of the nucleic acid sequence is configured toproduce one or more peptides that are displayed by an HLA class Imolecule. In some embodiments, the one or more peptides is a modified orunmodified fragment of a mutated KRAS protein. In some embodiments, themutated KRAS protein is selected from the group consisting of KRAS G12D,KRAS G12V, and KRAS G12R. In some embodiments, the nucleic acid sequenceis administered in an effective amount to a subject to prevent cancer.In some embodiments, the nucleic acid sequence is administered in aneffective amount to a subject to treat cancer.

In another aspect, the invention provides for an immunogenic peptidecomposition comprising two or more peptides selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.

In some embodiments, a peptide in the immunogenic peptide composition isdisplayed by an HLA class I molecule. In some embodiments, a peptide inthe immunogenic peptide composition is a modified or unmodified fragmentof a mutated KRAS protein. In some embodiments, the mutated KRAS proteinis selected from the group consisting of KRAS G12D, KRAS G12V, and KRASG12R. In some embodiments, the immunogenic peptide composition isadministered in an effective amount to a subject to prevent cancer. Insome embodiments, the immunogenic peptide composition is administered inan effective amount to a subject to treat cancer. In some embodiments,the immunogenic peptide composition comprises at least three peptidesselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.

In another aspect, the invention provides for a nucleic acid sequenceencoding one or more amino acid sequences selected from the groupconsisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:64, and SEQ ID NO: 65.

In some embodiments, the nucleic acid sequence is an immunogeniccomposition. In some embodiments, the nucleic acid sequence isadministered in a construct for expression in vivo. In some embodiments,the in vivo administration of the nucleic acid sequence is configured toproduce one or more peptides that are displayed by an HLA class IImolecule. In some embodiments, the one or more peptides is a modifiedfragment of a mutated KRAS protein. In some embodiments, the mutatedKRAS protein is selected from the group consisting of KRAS G12D, KRASG12V, KRAS G12R, KRAS G12C, and KRAS G13D. In some embodiments, thenucleic acid sequence is administered in an effective amount to asubject to prevent cancer. In some embodiments, the nucleic acidsequence is administered in an effective amount to a subject to treatcancer.

In another aspect, the invention provides for an immunogenic peptidecomposition comprising one or more peptides selected from the groupconsisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:64, and SEQ ID NO: 65.

In some embodiments, a peptide in the immunogenic peptide composition isdisplayed by an HLA class II molecule. In some embodiments, a peptide inthe immunogenic peptide composition is a modified or unmodified fragmentof a mutated KRAS protein. In some embodiments, the mutated KRAS proteinis selected from the group consisting of KRAS G12D, KRAS G12V, KRASG12R, KRAS G12C, and KRAS G13D. In some embodiments, the immunogenicpeptide composition is administered in an effective amount to a subjectto prevent cancer. In some embodiments, the immunogenic peptidecomposition is administered in an effective amount to a subject to treatcancer. In some embodiments, the immunogenic peptide compositioncomprises at least two peptides selected from the group consisting ofSEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ IDNO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60,SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ IDNO: 65.

Compositions

In some embodiments, the foreign peptides (e.g., peptide vaccine) areadministered in a pharmaceutical composition comprising the peptides anda pharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition is in the form of a spray, aerosol, gel,solution, emulsion, or suspension.

The composition is preferably administered to a subject with apharmaceutically acceptable carrier. Typically, in some embodiments, anappropriate amount of a pharmaceutically acceptable salt is used in theformulation, which in some embodiments can render the formulationisotonic.

In certain embodiments, the foreign peptides are provided as animmunogenic composition comprising any one of the foreign peptidesdescribed herein and a pharmaceutically acceptable carrier. In certainembodiments, the immunogenic composition further comprises an adjuvant.In certain embodiments, the foreign peptides are conjugated with othermolecules to increase their effectiveness as is known by those practicedin the art. For example, peptides can be coupled to antibodies thatrecognize cell surface proteins on antigen presenting cells to enhancevaccine effectiveness. One such method for increasing the effectivenessof peptide delivery is described in Woodham, et al. (2018).

In some embodiments, the pharmaceutically acceptable carrier is selectedfrom the group consisting of saline, Ringer's solution, dextrosesolution, and a combination thereof. Other suitable pharmaceuticallyacceptable carriers known in the art are contemplated. Suitable carriersand their formulations are described in Remington's PharmaceuticalSciences, 2005, Mack Publishing Co. The pH of the solution is preferablyfrom about 5 to about 8, and more preferably from about 7 to about 7.5.The formulation may also comprise a lyophilized powder. Further carriersinclude sustained release preparations such as semipermeable matrices ofsolid hydrophobic polymers, which matrices are in the form of shapedarticles, e.g., films, liposomes or microparticles. It will be apparentto those persons skilled in the art that certain carriers may be morepreferable depending upon, for instance, the route of administration andconcentration of peptides being administered.

The phrase pharmaceutically acceptable carrier as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subjectpharmaceutical agent from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier is acceptable in the senseof being compatible with the other ingredients of the formulation andnot injurious to the patient. Some examples of materials which can serveas pharmaceutically acceptable carriers include: sugars, such aslactose, glucose and sucrose; starches, such as corn starch and potatostarch; cellulose, and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients, such as cocoa butter and suppositorywaxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such as butyleneglycol; polyols, such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters, such as ethyl oleate and ethyl laurate; agar; bufferingagents, such as magnesium hydroxide and aluminum hydroxide; alginicacid; pyrogen-free water; isotonic saline; Ringer's solution; ethylalcohol; phosphate buffer solutions; and other non-toxic compatiblesubstances employed in pharmaceutical formulations. The term carrierdenotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing comingled with the compounds of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficiency. Thecomposition may also include additional agents such as an isotonicityagent, a preservative, a surfactant, and, a divalent cation, preferably,zinc.

The composition can also include an excipient, or an agent forstabilization of a foreign peptide composition, such as a buffer, areducing agent, a bulk protein, amino acids (such as e.g., glycine orpraline) or a carbohydrate. Bulk proteins useful in formulating foreignpeptide compositions include albumin. Typical carbohydrates useful informulating foreign peptides include but are not limited to sucrose,mannitol, lactose, trehalose, or glucose.

Surfactants may also be used to prevent soluble and insolubleaggregation and/or precipitation of foreign peptides or proteinsincluded in the composition. Suitable surfactants include but are notlimited to sorbitan trioleate, soya lecithin, and oleic acid. In certaincases, solution aerosols are preferred using solvents such as ethanol.Thus, formulations including foreign peptides can also include asurfactant that can reduce or prevent surface-induced aggregation offoreign peptides by atomization of the solution in forming an aerosol.Various conventional surfactants can be employed, such aspolyoxyethylene fatty acid esters and alcohols, and polyoxyethylenesorbitol fatty acid esters. Amounts will generally range between 0.001%and 4% by weight of the formulation. In some embodiments, surfactantsused with the present disclosure are polyoxyethylene sorbitanmono-oleate, polysorbate 80, polysorbate 20. Additional agents known inthe art can also be included in the composition.

In some embodiments, the pharmaceutical compositions and dosage formsfurther comprise one or more compounds that reduce the rate by which anactive ingredient will decay, or the composition will change incharacter. So called stabilizers or preservatives may include, but arenot limited to, amino acids, antioxidants, pH buffers, or salt buffers.Nonlimiting examples of antioxidants include butylated hydroxy anisole(BHA), ascorbic acid and derivatives thereof, tocopherol and derivativesthereof, butylated hydroxy anisole and cysteine. Nonlimiting examples ofpreservatives include parabens, such as methyl or propylp-hydroxybenzoate and benzalkonium chloride. Additional nonlimitingexamples of amino acids include glycine or proline.

The present invention also teaches the stabilization (preventing orminimizing thermally or mechanically induced soluble or insolubleaggregation and/or precipitation of an inhibitor protein) of liquidsolutions containing foreign peptides at neutral pH or less than neutralpH by the use of amino acids including proline or glycine, with orwithout divalent cations resulting in clear or nearly clear solutionsthat are stable at room temperature or preferred for pharmaceuticaladministration.

In one embodiment, the composition is a pharmaceutical composition ofsingle unit or multiple unit dosage forms. Pharmaceutical compositionsof single unit or multiple unit dosage forms of the invention comprise aprophylactically or therapeutically effective amount of one or morecompositions (e.g., a compound of the invention, or other prophylacticor therapeutic agent), typically, one or more vehicles, carriers, orexcipients, stabilizing agents, and/or preservatives. Preferably, thevehicles, carriers, excipients, stabilizing agents and preservatives arepharmaceutically acceptable.

In some embodiments, the pharmaceutical compositions and dosage formscomprise anhydrous pharmaceutical compositions and dosage forms.Anhydrous pharmaceutical compositions and dosage forms of the inventioncan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprise a primary or secondary amine are preferablyanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected. An anhydrouspharmaceutical composition should be prepared and stored such that itsanhydrous nature is maintained. Accordingly, anhydrous compositions arepreferably packaged using materials known to prevent exposure to watersuch that they can be included in suitable formulary kits. Examples ofsuitable packaging include, but are not limited to, hermetically sealedfoils, plastics, unit dose containers (e.g., vials), blister packs, andstrip packs.

Suitable vehicles are well known to those skilled in the art ofpharmacy, and non-limiting examples of suitable vehicles includeglucose, sucrose, starch, lactose, gelatin, rice, silica gel, glycerol,talc, sodium chloride, dried skim milk, propylene glycol, water, sodiumstearate, ethanol, and similar substances well known in the art. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid vehicles. Whether a particular vehicle is suitablefor incorporation into a pharmaceutical composition or dosage formdepends on a variety of factors well known in the art including, but notlimited to, the way in which the dosage form will be administered to apatient and the specific active ingredients in the dosage form.Pharmaceutical vehicles can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.

The invention also provides that a pharmaceutical composition can bepackaged in a hermetically sealed container such as an ampoule orsachette indicating the quantity. In one embodiment, the pharmaceuticalcomposition can be supplied as a dry sterilized lyophilized powder in adelivery device suitable for administration to the lower airways of apatient. The pharmaceutical compositions can, if desired, be presentedin a pack or dispenser device that can contain one or more unit dosageforms containing the active ingredient. The pack can for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device can be accompanied by instructions for administration.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for administration may be in theform of powders, granules, or as a solution or a suspension in anaqueous or non-aqueous liquid, or as an oil-in-water or water-in-oilliquid emulsion, or as an elixir or syrup, or as pastilles (using aninert base, such as gelatin and glycerin, or sucrose and acacia) and/oras mouthwashes and the like, each containing a predetermined amount of acompound of the present invention (e.g., peptides) as an activeingredient.

A liquid composition herein can be used as such with a delivery device,or they can be used for the preparation of pharmaceutically acceptableformulations comprising foreign peptides that are prepared for exampleby the method of spray drying. The methods of spray freeze-dryingforeign peptides/proteins for pharmaceutical administration disclosed inMaa et al., Curr. Pharm. Biotechnol., 2001, 1, 283-302, are incorporatedherein. In another embodiment, the liquid solutions herein are freezespray dried and the spray-dried product is collected as a dispersibleforeign peptide-containing powder that is therapeutically effective whenadministered to an individual.

The compounds and pharmaceutical compositions of the present inventioncan be employed in combination therapies, that is, the compounds andpharmaceutical compositions can be administered concurrently with, priorto, or subsequent to, one or more other desired therapeutics or medicalprocedures (e.g., foreign peptide vaccine can be used in combinationtherapy with another treatment such as chemotherapy, radiation,pharmaceutical agents, and/or another treatment). The particularcombination of therapies (therapeutics or procedures) to employ in acombination regimen will take into account compatibility of the desiredtherapeutics and/or procedures and the desired therapeutic effect to beachieved. It will also be appreciated that the therapies employed mayachieve a desired effect for the same disorder (for example, thecompound of the present invention may be administered concurrently withanother therapeutic or prophylactic).

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

The current invention provides for dosage forms comprising foreignpeptides suitable for treating cancer or other diseases. The dosageforms can be formulated, e.g., as sprays, aerosols, nanoparticles,liposomes, or other forms known to one of skill in the art. See, e.g.,Remington's Pharmaceutical Sciences; Remington: The Science and Practiceof Pharmacy supra; Pharmaceutical Dosage Forms and Drug Delivery Systemsby Howard C., Ansel et al., Lippincott Williams & Wilkins; 7th edition(Oct. 1, 1999).

Generally, a dosage form used in the acute treatment of a disease maycontain larger amounts of one or more of the active ingredients itcomprises than a dosage form used in the chronic treatment of the samedisease. In addition, the prophylactically and therapeutically effectivedosage form may vary among different conditions. For example, atherapeutically effective dosage form may contain peptides that has anappropriate immunogenic action when intending to treat cancer or otherdisease. On the other hand, a different effective dosage may containforeign peptides that has an appropriate immunogenic action whenintending to use the peptides of the invention as a prophylactic (e.g.,vaccine) against cancer or another disease/condition. These and otherways in which specific dosage forms encompassed by this invention willvary from one another and will be readily apparent to those skilled inthe art. See, e.g., Remington's Pharmaceutical Sciences, 2005, MackPublishing Co.; Remington: The Science and Practice of Pharmacy byGennaro, Lippincott Williams & Wilkins; 20th edition (2003);Pharmaceutical Dosage Forms and Drug Delivery Systems by Howard C. Anselet al., Lippincott Williams & Wilkins; 7th edition (Oct. 1, 1999); andEncyclopedia of Pharmaceutical Technology, edited by Swarbrick, J. & J.C. Boylan, Marcel Dekker, Inc., New York, 1988, which are incorporatedherein by reference in their entirety.

The pH of a pharmaceutical composition or dosage form may also beadjusted to improve delivery and/or stability of one or more activeingredients. Similarly, the polarity of a solvent carrier, its ionicstrength, or tonicity can be adjusted to improve delivery. Compoundssuch as stearates can also be added to pharmaceutical compositions ordosage forms to alter advantageously the hydrophilicity or lipophilicityof one or more active ingredients to improve delivery. In this regard,stearates can also serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery enhancing orpenetration-enhancing agent. Different salts, hydrates, or solvates ofthe active ingredients can be used to adjust further the properties ofthe resulting composition.

Compositions can be formulated with appropriate carriers and adjuvantsusing techniques to yield compositions suitable for immunization. Thecompositions can include an adjuvant, such as, for example but notlimited to, alum, poly IC, MF-59, squalene-based adjuvants, or liposomalbased adjuvants suitable for immunization.

In some embodiments, the compositions and methods comprise any suitableagent or immune modulation which could modulate mechanisms of hostimmune tolerance and release of the induced antibodies. In certainembodiments, an immunomodulatory agent is administered in at time and inan amount sufficient for transient modulation of the subject's immuneresponse so as to induce an immune response which comprises antibodiesagainst for example tumor neoantigens (i.e., tumor-specific antigens(TSA)).

Expression Systems

In certain aspects, the invention provides culturing a cell line thatexpresses any one of the foreign peptides of the invention in a culturemedium comprising any of the foreign peptides described herein.

Various expression systems for producing recombinant proteins/peptidesare known in the art, and include, prokaryotic (e.g., bacteria), plant,insect, yeast, and mammalian expression systems. Suitable cell lines,can be transformed, transduced, or transfected with nucleic acidscontaining coding sequences for the foreign peptides of the invention inorder to produce the molecule of interest. Expression vectors containingsuch a nucleic acid sequence, which can be linked to at least oneregulatory sequence in a manner that allows expression of the nucleotidesequence in a host cell, can be introduced via methods known in the art.Practitioners in the art understand that designing an expression vectorcan depend on factors, such as the choice of host cell to be transfectedand/or the type and/or amount of desired protein to be expressed.Enhancer regions, which are those sequences found upstream or downstreamof the promoter region in non-coding DNA regions, are also known in theart to be important in optimizing expression. If needed, origins ofreplication from viral sources can be employed, such as if a prokaryotichost is utilized for introduction of plasmid DNA. However, in eukaryoticorganisms, chromosome integration is a common mechanism for DNAreplication. For stable transfection of mammalian cells, a smallfraction of cells can integrate introduced DNA into their genomes. Theexpression vector and transfection method utilized can be factors thatcontribute to a successful integration event. For stable amplificationand expression of a desired protein, a vector containing DNA encoding aprotein of interest is stably integrated into the genome of eukaryoticcells (for example mammalian cells), resulting in the stable expressionof transfected genes. A gene that encodes a selectable marker (forexample, resistance to antibiotics or drugs) can be introduced into hostcells along with the gene of interest in order to identify and selectclones that stably express a gene encoding a protein of interest. Cellscontaining the gene of interest can be identified by drug selectionwherein cells that have incorporated the selectable marker gene willsurvive in the presence of the drug. Cells that have not incorporatedthe gene for the selectable marker die. Surviving cells can then bescreened for the production of the desired protein molecule.

A host cell strain, which modulates the expression of the insertedsequences, or modifies and processes the nucleic acid in a specificfashion desired also may be chosen. Such modifications (for example,glycosylation and other post-translational modifications) and processing(for example, cleavage) of peptide/protein products may be important forthe function of the peptide/protein. Different host cell strains havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. As such,appropriate host systems or cell lines can be chosen to ensure thecorrect modification and processing of the foreign protein expressed.Thus, eukaryotic host cells possessing the cellular machinery for properprocessing of the primary transcript, glycosylation, and phosphorylationof the gene product may be used.

Various culturing parameters can be used with respect to the host cellbeing cultured. Appropriate culture conditions for mammalian cells arewell known in the art (Cleveland W L, et al., J Immunol Methods, 1983,56(2): 221-234) or can be determined by the skilled artisan (see, forexample, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D.and Hames, B. D., eds. (Oxford University Press: New York, 1992)). Cellculturing conditions can vary according to the type of host cellselected. Commercially available medium can be utilized.

Foreign peptides of the invention can be purified from any human ornon-human cell which expresses the polypeptide, including those whichhave been transfected with expression constructs that express foreignpeptides of the invention. For protein recovery, isolation and/orpurification, the cell culture medium or cell lysate is centrifuged toremove particulate cells and cell debris. The desired polypeptidemolecule is isolated or purified away from contaminating solubleproteins and polypeptides by suitable purification techniques.Non-limiting purification methods for proteins include: size exclusionchromatography; affinity chromatography; ion exchange chromatography;ethanol precipitation; reverse phase HPLC; chromatography on a resin,such as silica, or cation exchange resin, e.g., DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, e.g.,Sephadex G-75, Sepharose; protein A sepharose chromatography for removalof immunoglobulin contaminants; and the like. Other additives, such asprotease inhibitors (e.g., PMSF or proteinase K) can be used to inhibitproteolytic degradation during purification. Purification proceduresthat can select for carbohydrates can also be used, e.g., ion-exchangesoft gel chromatography, or HPLC using cation- or anionexchange resins,in which the more acidic fraction(s) is/are collected.

Methods of Treatment

In one embodiment, the subject matter disclosed herein relates to apreventive medical treatment started after following diagnosis of cancerin order to prevent the disease from worsening or curing the disease. Inone embodiment, the subject matter disclosed herein relates toprophylaxis of subjects who are believed to be at risk for cancer orhave previously been diagnosed with cancer (or another disease). In oneembodiment, said subjects can be administered the peptide vaccinedescribed herein or pharmaceutical compositions thereof. The inventioncontemplates using any of the foreign peptides produced by the systemsand methods described herein. In one embodiment, the foreign peptidevaccines described herein can be administered subcutaneously via syringeor any other suitable method know in the art.

The compound(s) or combination of compounds disclosed herein, orpharmaceutical compositions may be administered to a cell, mammal, orhuman by any suitable means. Non-limiting examples of methods ofadministration include, among others, (a) administration though oralpathways, which includes administration in capsule, tablet, granule,spray, syrup, or other such forms; (b) administration through non-oralpathways such as intraocular, intranasal, intraauricular, rectal,vaginal, intraurethral, transmucosal, buccal, or transdermal, whichincludes administration as an aqueous suspension, an oily preparation orthe like or as a drip, spray, suppository, salve, ointment or the like;(c) administration via injection, including subcutaneously,intraperitoneally, intravenously, intramuscularly, intradermally,intraorbitally, intracapsularly, intraspinally, intrasternally, or thelike, including infusion pump delivery; (d) administration locally suchas by injection directly in the renal or cardiac area, e.g., by depotimplantation; (e) administration topically; as deemed appropriate bythose of skill in the art for bringing the compound or combination ofcompounds disclosed herein into contact with living tissue; (f)administration via inhalation, including through aerosolized, nebulized,and powdered formulations; and (g) administration through implantation.

As will be readily apparent to one skilled in the art, the effective invivo dose to be administered and the particular mode of administrationwill vary depending upon the age, weight and species treated, and thespecific use for which the compound or combination of compoundsdisclosed herein are employed. The determination of effective doselevels, that is the dose levels necessary to achieve the desired result,can be accomplished by one skilled in the art using routinepharmacological methods. Typically, human clinical applications ofproducts are commenced at lower dose levels, with dose level beingincreased until the desired effect is achieved. Alternatively,acceptable in vitro studies can be used to establish useful doses androutes of administration of the compositions identified by the presentmethods using established pharmacological methods. Effective animaldoses from in vivo studies can be converted to appropriate human dosesusing conversion methods known in the art (e.g., see Nair A B, Jacob S.A simple practice guide for dose conversion between animals and human.Journal of basic and clinical pharmacy. 2016 March; 7(2):27.)

Methods of Prevention

In some embodiments, the foreign peptides prepared using methods of theinvention can be used as a vaccine to promote an immune response againstcancer (e.g., against tumor neoantigens). In some embodiments, theinvention provides compositions and methods for induction of immuneresponse, for example induction of antibodies to tumor neoantigens. Insome embodiments, the antibodies are broadly neutralizing antibodies. Insome embodiments, the foreign peptides prepared using methods of theinvention can be used as a vaccine to promote an immune response againsta pathogen.

The compositions, systems, and methods disclosed herein are not to belimited in scope to the specific embodiments described herein. Indeed,various modifications of the compositions, systems, and methods inaddition to those described will become apparent to those of skill inthe art from the foregoing description.

What is claimed is:
 1. A composition comprising nucleic acid sequencesencoding two or more amino acid sequences selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, and SEQ ID NO:
 41. 2. The composition of claim 1,wherein the composition is immunogenic.
 3. The composition of claim 1,wherein the nucleic acid sequence is administered in a construct forexpression in vivo.
 4. The composition of claim 3, wherein the in vivoadministration of the nucleic acid sequence is configured to produce oneor more peptides that are displayed by an HLA class I molecule.
 5. Thecomposition of claim 4, wherein the one or more peptides is a modifiedor unmodified fragment of a mutated KRAS protein.
 6. The composition ofclaim 5, wherein the mutated KRAS protein is selected from the groupconsisting of KRAS G12D, KRAS G12V, and KRAS G12R.
 7. The composition ofclaim 3, wherein the nucleic acid sequence is administered in aneffective amount to a subject to prevent cancer.
 8. The composition ofclaim 3, wherein the nucleic acid sequence is administered in aneffective amount to a subject to treat cancer.
 9. An immunogenic peptidecomposition comprising two or more peptides selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, and SEQ ID NO:
 41. 10. The immunogenic peptidecomposition of claim 9, wherein a peptide in the immunogenic peptidecomposition is displayed by an HLA class I molecule.
 11. The immunogenicpeptide composition of claim 9, wherein a peptide in the immunogenicpeptide composition is a modified or unmodified fragment of a mutatedKRAS protein.
 12. The immunogenic peptide composition of claim 11,wherein the mutated KRAS protein is selected from the group consistingof KRAS G12D, KRAS G12V, and KRAS G12R.
 13. The immunogenic peptidecomposition of claim 9, wherein the immunogenic peptide composition isadministered in an effective amount to a subject to prevent cancer. 14.The immunogenic peptide composition of claim 9, wherein the immunogenicpeptide composition is administered in an effective amount to a subjectto treat cancer.
 15. The immunogenic peptide composition of claim 9,wherein the immunogenic peptide composition comprises at least threepeptides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ IDNO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.16. A nucleic acid sequence encoding one or more amino acid sequencesselected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48,SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ IDNO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQID NO: 63, SEQ ID NO: 64, and SEQ ID NO:
 65. 17. The nucleic acidsequence of claim 16, wherein the nucleic acid sequence is animmunogenic composition.
 18. The nucleic acid sequence of claim 16,wherein the nucleic acid sequence is administered in a construct forexpression in vivo.
 19. The nucleic acid sequence of claim 18, whereinthe in vivo administration of the nucleic acid sequence is configured toproduce one or more peptides that are displayed by an HLA class IImolecule.
 20. The nucleic acid sequence of claim 16, wherein the one ormore peptides is a modified fragment of a mutated KRAS protein.
 21. Thenucleic acid sequence of claim 19, wherein the mutated KRAS protein isselected from the group consisting of KRAS G12D, KRAS G12V, KRAS G12R,KRAS G12C, and KRAS G13D.
 22. The nucleic acid sequence of claim 16,wherein the nucleic acid sequence is administered in an effective amountto a subject to prevent cancer.
 23. The nucleic acid sequence of claim16, wherein the nucleic acid sequence is administered in an effectiveamount to a subject to treat cancer.
 24. An immunogenic peptidecomposition comprising one or more peptides selected from the groupconsisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:64, and SEQ ID NO:
 65. 25. The immunogenic peptide composition of claim24, wherein a peptide in the immunogenic peptide composition isdisplayed by an HLA class II molecule.
 26. The immunogenic peptidecomposition of claim 24, wherein a peptide in the immunogenic peptidecomposition is a modified or unmodified fragment of a mutated KRASprotein.
 27. The immunogenic peptide composition of claim 26, whereinthe mutated KRAS protein is selected from the group consisting of KRASG12D, KRAS G12V, KRAS G12R, KRAS G12C, and KRAS G13D.
 28. Theimmunogenic peptide composition of claim 24, wherein the immunogenicpeptide composition is administered in an effective amount to a subjectto prevent cancer.
 29. The immunogenic peptide composition of claim 24,wherein the immunogenic peptide composition is administered in aneffective amount to a subject to treat cancer.
 30. The immunogenicpeptide composition of claim 24, wherein the immunogenic peptidecomposition comprises at least two peptides selected from the groupconsisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO:64, and SEQ ID NO: 65.